Gene, 1997 Oct 1, 198(1-2), 43 - 52 The ER chaperone encoding bipA gene of black Aspergilli is induced by heat shock and unfolded proteins; van Gemeren IA et al.; We describe the cloning and characterisation of the BiP gene homologues of the filamentous fungi Aspergillus niger and Aspergillus awamori . The BiP genes of these black Aspergilli encode an identical protein of 672 amino acids, which has a high homology with the BiP protein from Saccharomyces cerevisiae and contains a putative signal sequence of 38 amino acids . The DNA sequences of the Aspergillus BiP genes diverge in particular in the three intronic sequences and the 5'- and 3'- noncoding regions . Sequences resembling Heat Shock Elements (HSE) and Unfolded Protein Response (UPR) elements, as found in the yeast KAR2 promoter, are present in the 5' non-transcribed regions of both genes . The expression of the A . niger bipA gene is increased by heat shock and tunicamycin treatment. FEBS Lett, 1997 Oct 20, 416(2), 135 - 8 The different inhibitory domains of the Oct-2 transcription factor have distinct functional activities; Gay RD et al.; The Oct-2 POU family transcription factor contains three distinct regions whose deletion reduces its ability to inhibit transcription via its octamer binding site . Here we show that only one of these inhibitory domains is capable of also inhibiting the activity of activating molecules bound at adjacent sites upstream of a TATA box-containing promoter whereas the other two regions are inactive in this assay . None of the three regions is able to achieve this effect when located upstream of the same promoter containing an initiator motif . The mechanisms of action of these domains and their role in the functioning of the Oct-2 factor are discussed. Biochem Biophys Res Commun, 1997 Nov 7, 240(1), 228 - 33 The trimerization domain of human heat shock factor 2 is able to interact with nucleoporin p62; Yoshima T et al.; Heat shock factor 2 (HSF2) acquires DNA binding activity during hemin-induced differentiation of human K562 erythroleukemia cells . To investigate the mechanisms responsible for the regulation of HSF2 activity, we searched for proteins that can associate with HSF2 by the yeast two-hybrid system . Nucleoporin p62, a major component of the nuclear pore complex, was cloned from cDNA libraries of K562 cells . We demonstrated physical interaction between HSF2 and p62 both by a glutathione S-transferase (GST) pull-down assay in vitro and by a two-hybrid assay in K562 cells . HSF1 is also able to interact with p62 on a GST pull-down assay, but not on a mammalian two-hybrid system . Furthermore, it was shown that this interaction occurred between the trimerization domain of HSF2 and the C-terminal alpha-helical coiled-coil domain of p62 . These data suggest the possibility that p62 is involved in the activation or regulation of HSF2. Biochem Biophys Res Commun, 1997 Oct 29, 239(3), 710 - 4 Overproduction of Mpd2p suppresses the lethality of protein disulfide isomerase depletion in a CXXC sequence dependent manner; Tachikawa H et al.; The third multicopy suppressor gene of the PDI1 deletion from Saccharomyces cerevisiae, MPD2, was isolated and characterized . The MPD2 gene encodes a protein with a putative signal sequence, ER retention signal, and a disulfide isomerase active site like sequence . The amino acid sequence around the active site like sequence is similar to the thioredoxin-like domains of PDI and PDI related proteins, although the similarity is comparatively low . A delta-pdi1 strain over-producing Mpd2p showed slow growth and was sensitive to 1 mM dithiothreitol . Mpd2p can be detected in wild type cells and is a glycoprotein . Although the MPD2 gene was not essential for growth, overexpression of the gene partially restored the maturation defect of carboxypeptidase Y caused by the PDI1 deletion . Mutagenesis analysis revealed that Mpd2p can compensate for the loss of PDI with its CXXC sequence. J Mol Biol, 1997 Oct 17, 273(1), 1 - 6 A mutant form of mitochondrial GrpE suppresses the sorting defect caused by an alteration in the presequence of cytochrome b2; Merlin A et al.; Transport of preproteins across the inner mitochondrial membrane requires the action of the matrix heat shock protein Hsp70 . Together with its co-chaperone mitochondrial GrpE (Mge1), mtHsp70 transiently binds to the inner membrane translocase subunit Tim44 in a nucleotide-regulated manner, forming an ATP-dependent import driving machinery . We report that a mutant form of Mge1 (Mge1-100) is completely absent in mtHsp70-Tim44 complexes, although its ability to interact with soluble mtHsp70 is only partially reduced . While this mge1-100 mutation only partially retards preprotein translocation into the matrix, it exerts a selective effect on sorting of cytochrome b2 to the intermembrane space . A cytochrome b2 with an altered sorting signal, which is only processed to the intermediate stage and mistargeted to the matrix of wild-type mitochondria, is processed to the mature form and correctly targeted to the intermembrane space of mge1-100 mitochondria . These results suggest that (1) Mge1-100 discriminates between soluble and membrane-bound mtHsp70 and (2) the membrane-bound mtHsp70-Mge1 driving system competes with the sorting machinery for translocation of preproteins like cytochrome b2. J Invertebr Pathol, 1997 Nov, 70(3), 226 - 33 Heat-shock response in a molluscan cell line: characterization of the response and cloning of an inducible HSP70 cDNA; Laursen JR et al.; Sublethal heat-shock of cells of the Bge (Biomphalaria glabrata embryonic) snail cell line resulted in increased or new expression of metabolically labeled polypeptides of approximately 21.5, 41, 70, and 74 kDa molecular mass . Regulation of this response appeared to be at the transcriptional level since a similar protein banding pattern was seen upon SDS-PAGE/fluorographic analysis of polypeptides produced by in vitro translation of total RNA from cells subjected to heat shock . Using a yeast (Saccharomyces cerevisiae) 70-kDa heat-shock protein (HSP70) probe to screen a cDNA library from heat-treated Bge cells, we isolated a full-length cDNA clone encoding a putative Bge HSP70 . The cDNA was 2453 bp in length and contained an open reading frame of 1908 bp encoding a 636-amino-acid polypeptide with calculated molecular mass of 70,740 Da . Comparison of a conserved region of 209 amino acid residues revealed > 80% identity between the deduced amino acid sequence of Bge HSP70 and that of yeast (81%), the human blood fluke Schistosoma mansoni (for which B . glabrata serves as intermediate host) (81%), Drosophila (81%), human (84%), and the marine gastropod Aplysia californica (88%, 90%) . In addition to the extensive sharing of sequence homology, the identification of several eukaryotic HSP70 signature sequences and an N-linked glycosylation site characteristic of cytoplasmic HSPs strongly support the identity of the Bge cDNA as encoding an authentic HSP70 . Results of a Northern blot analysis, using Bge HSP70 clone-specific probes, indicated that gene expression was heat inducible and not constitutively expressed . This is the first reported sequence of an inducible HSP70 from cells originating from a freshwater gastropod and provides a first step in the development of a genetic transformation system for molluscs of medical importance. J Biol Chem, 1997 Nov 14, 272(46), 29372 - 9 Activation of the Ste20-like oxidant stress response kinase-1 during the initial stages of chemical anoxia-induced necrotic cell death . Requirement for dual inputs of oxidant stress and increased cytosolic {Ca2+}; Pombo CM et al.; Signal transduction mechanisms activated during the early stages of necrotic cell death are poorly characterized . We have recently identified the Sterile 20 (Ste20)-like oxidant stress response kinase-1, SOK-1, which is a member of the Ste20 kinase family . We report that SOK-1 is markedly activated as early as 20 min after chemical anoxia induced by exposure of Madin-Darby canine kidney or LLC-PK1 renal tubular epithelial cells to 2-deoxyglucose (2-DG) and any one of three inhibitors of the electron transport chain, cyanide (CN), rotenone, or antimycin A . Since oxidant stress activates SOK-1, we postulated that reactive oxygen species (ROS), which are produced by the electron transport chain during chemical anoxia, might be responsible for SOK-1 activation . The time course of CN/2-DG-induced SOK-1 activation and of production of ROS, measured in cells loaded with dichlorofluorescein, were compatible with a role for ROS in SOK-1 activation . Furthermore, preincubation of LLC-PK1 cells with three unrelated scavengers of ROS, pyrrolidine dithiocarbamate, pyruvate, or nordihydroguaiaretic acid, reduced both cellular oxidant stress and activation of SOK-1 by CN/2-DG . An increase in cytosolic free {Ca2+} ({Ca2+}i) was necessary but not sufficient for CN/2-DG-induced activation of SOK-1 . Preincubation of cells with BAPTA-AM prevented activation of SOK-1 . Incubation of cells with thapsigargin or the calcium ionophore, A23187, had no effect on SOK-1 activity, but preincubation of cells with either of these agents markedly enhanced CN/2-DG-induced activation of SOK-1 (20-fold versus 7-fold) . In summary, chemical anoxia activates SOK-1 via an oxidant stress-dependent mechanism that is both critically dependent upon and markedly amplified by an increase in {Ca2+}i . This requirement for dual inputs of oxidant stress and an increase in {Ca2+}i may prevent inappropriate activation of the kinase by milder degrees of oxidant stress, which are insufficient to generate an increase in {Ca2+}i . The activation of SOK-1 may be one of the cell's earliest responses to inducers of necrotic cell death. J Biol Chem, 1997 Nov 14, 272(46), 29200 - 6 The coatomer protein beta'-COP, a selective binding protein (RACK) for protein kinase Cepsilon; Csukai M et al.; Distinct subcellular localization of activated protein kinase C (PKC) isozymes is mediated by their binding to isozyme-specific RACKs (receptors for activated C-kinase) . Our laboratory has previously isolated one such protein, RACK1, and demonstrated that this protein displays specificity for PKCbeta . We have recently shown that at least part of the PKCepsilon RACK-binding site on PKCepsilon lies within the unique V1 region of this isozyme (Johnson, J . A., Gray, M . O., Chen, C.-H., and Mochly-Rosen, D . (1996) J . Biol . Chem . 271, 24962-24966) . Here, we have used the PKCepsilon V1 region to clone a PKCepsilon-selective RACK, which was identified as the COPI coatomer protein, beta'-COP . Similar to RACK1, beta'-COP contains seven repeats of the WD40 motif and fulfills the criteria previously established for RACKs . Activated PKCepsilon colocalizes with beta'-COP in cardiac myocytes and binds to Golgi membranes in a beta'-COP-dependent manner . A role for PKC in control of secretion has been previously suggested, but this is the first report of direct protein/protein interaction of PKCepsilon with a protein involved in vesicular trafficking. Eur J Biochem, 1997 Oct 1, 249(1), 239 - 47 Human and murine serine-palmitoyl-CoA transferase--cloning, expression and characterization of the key enzyme in sphingolipid synthesis; Weiss B et al.; Serine palmitoyltransferase (SPT, EC 2.3.1.50) is the key enzyme in sphingolipid biosynthesis . It catalyzes the pyridoxal-5'-phosphate-dependent condensation of L-serine and palmitoyl-CoA to 3-oxosphinganine . Human expressed-sequence-tag (EST) clones are similar to the two yeast genes for synthesis of long-chain bases, LCB1 and LCB2, which are believed to encode two subunits of SPT {Buede, R., Pinto, W . J., Lester, R . L . & Dickson, R . C . (1991) J . Bacteriol . 173, 4325-5332; Nagiec, M . M., Baltisberger, J . A., Wells, G . B., Lester, R . L . & Dickson, R . C . (1994) Proc . Natl Acad . Sci . USA 91, 7899-7902} . We have cloned and characterized two complete human and murine cDNA sequences named hLCB1 & mLCB1 and hLCB2 & mLCB2, respectively, similar to the yeast LCB1 and LCB2 genes . Human embryonic kidney cells (HEK 293) transfected with murine sequences of LCB1 (mLCB1) and LCB2 (mLCB2) independently and in coexpression showed an overexpression of the transcripts on the mRNA and protein level . The enzymatic activity of cells expressing mLCB2 alone or coexpressed with mLCB1 was three times higher than the activity of untransfected HEK cells . mLCB1 expression was not required for the synthesis of 3-oxo-sphinganine in mammalian cells . Transcription/translation in vitro yielded mLCB1 (53 kDa) and mLCB2 (63 kDa) . The two proteins do not contain a signal peptide nor are they glycosylated . The endogenous and overexpressed SPT activity were both sensitive to common SPT inhibitors . Labeling studies with {1-(14)C}palmitic acid indicated that cell lines transfected with mLCB2 preferentially use the excess sphingoid bases for glucocerebroside and galactocerebroside synthesis . Our results provide conclusive genetic and biochemical evidence that the human and murine LCB2 genes described here encode serine palmitoyltransferase . Further studies will be required to unravel the function of the LCB1 gene in mammalian cells. Eur J Biochem, 1997 Oct 1, 249(1), 142 - 8 Peptide fragments of DNA topoisomerase II with helix-forming and coiled-coil-forming properties act as inhibitors of the enzyme; Frere-Gallois V et al.; We have previously shown that a synthetic peptide (dL) consisting of amino acids 1013-1056 of human alpha topoisomerase II adopted an alpha-helix structure and formed a stable dimer coiled-coil in solution {Frere, V., Sourgen . F., Monnot, M., Troalen, F . & Fermandjian, S . (1995) J . Biol . Chem . 270, 17502-17507} . Here we studied two peptides, dP and dLshort, which are related to dL but which have a double substitution Leu1026-->Pro, Leu1037-->Pro and a deletion of the 15 C-terminal residues, respectively . The peptides were studied for their ability to form alpha-helix structures, coiled coils, and to inhibit topoisomerase II activity . In combining circular dichroism spectra with AGADIR prediction for helix structures, we demonstrated that the dLshort peptide, like its parent dL peptide, adopts an alpha-helix structure and can autoassociate into coiled-coils, while dP is completely devoid of such properties . Remarkably, only the dL and dLshort peptides act as good inhibitors of topoisomerase II in various in vitro assays . However, the dLshort peptide has a stronger helix potential and behaves as a much more potent inhibitor (5 microM versus 200 microM) compared to the dL peptide . All these data strongly suggest that the greater inhibitory effect demonstrated by the dLshort peptide is related to its higher ability to form a stable amphiphilic helix, which in turn better recognizes its homologous helical segment in topoisomerase II . Finally, we propose that the dL and the dLshort peptides could interfere with the enzymatic activity of topoisomersase II in modifying its autoassociation or translocation properties . Such peptides may serve as useful models for developing simpler and more specific inhibitors of topoisomerase II. Eur J Biochem, 1997 Oct 1, 249(1), 85 - 91 The BH3 domain of Bax is sufficient for interaction of Bax with itself and with other family members and it is required for induction of apoptosis; Simonen M et al.; bax is an apoptosis-inducing member of the bcl-2 multigene family . We have studied interactions of human Bax with itself, and with the apoptosis-preventing members Bcl-2 and Bcl-xL using a yeast two-hybrid system . Exhaustive Bax truncations were constructed and their interactions with full-length family members studied . Bax interacted similarly with itself as with the apoptosis-suppressing family members Bcl-2 and Bcl-xL in quantitative two-hybrid studies . A region of 41 amino acids covering the recently discovered BH3 domain of Bax was found to be necessary and sufficient for all interactions of Bax . Bax truncations containing BH3, but lacking BH1 and BH2 homology domains, interacted with the other family members markedly more strongly than full-length Bax, which may reflect conformational changes required for the interactions of full-length Bax . The minimum requirements for Bax homodimerization were found to be the BH3 domain from one Bax molecule and a region covering BH3 plus BH1 from another . We also studied the apoptosis-inducing activity of the Bax truncations upon microinjection of expression plasmids into rat fibroblasts . The BH3 region was required for the apoptosis-inducing activity of Bax, whereas BH1, BH2 and the N-terminus of Bax were dispensable. J Biol Chem, 1997 Nov 7, 272(45), 28198 - 201 Preferential interaction of sentrin with a ubiquitin-conjugating enzyme, Ubc9; Gong L et al.; Sentrin is a ubiquitin-like molecule that has been shown to interact with the death domains of Fas and tumor necrosis factor receptor 1 (TNFR1), PML, Rad51, Rad52, and RanGAP1 . We have reported previously that sentrin can be conjugated to other proteins in a manner analogous to protein ubiquitination (Kamitani, T., Nguyen, H . P., and Yeh, E . T . H . (1997) J . Biol . Chem . 272, 14001-14004) . Furthermore, the conserved C-terminal Gly-Gly residues are required for sentrinization to occur . To identify enzymes which play a role in sentrinization, the yeast two-hybrid system was used to screen a human placenta cDNA library using sentrin as bait . A strong positive interacting clone was found to contain a cDNA insert encoding the ubiquitin-conjugating enzyme, Ubc9 . The interaction between sentrin and Ubc9 required the ubiquitin domain and the C-terminal Gly-Gly residues of sentrin . This interaction appears to be specific because sentrin could only interact weakly with UbcH5B, but could not interact with HHR6B, UbcH6 nor E2-EPF . In vitro translated sentrin could be precipitated by a GST-Ubc9 fusion protein, but not by glutathione S-transferase . A beta-mercaptoethanol-sensitive Ubc9-sentrin conjugate could also be identified in the in vitro binding assay . Substitution of the conserved cysteine residue of Ubc9 by serine abolished the formation of the Ubc9-sentrin conjugate . Taken together, Ubc9 is a strong candidate to be the key conjugating enzyme in the sentrinization pathway. Biochemistry (Mosc), 1997 Aug, 62(8), 850 - 7 Specific cleavage of hybrid proteins by proteinase encoded by the KEX2 gene; Bessmertnaya LYa et al.; A method for isolation of the KEX2-gene-encoded membrane-bound proteinase from alpha-cells of Saccharomyces cerevisiae yeast has been modified . The isolated enzyme hydrolyzes peptides and proteins with basic amino acid pairs which are cleaved at the C-ends of their peptide bonds . Because KEX2 proteinase is located within the Golgi compartment, it may be isolated by differential centrifugation of broken cells at 7000g for 15 min and at 20,000g for 15 min . By extracting the fraction that contains the active enzyme by a detergent solution, a protein has been obtained with specific activity 30 times higher than that of the membrane extract prepared according to the standard technique . This protocol decreases the number of steps required to isolate the enzyme . The effects of pH and inhibitors on KEX2 proteinase-catalyzed hydrolysis of Ac-Leu-Lys-Arg-pNA were studied . KEX2 proteinase can participate in peptide hormone processing because it cleaves human proinsulin at the peptide bond between Arg32 and Glu33 . The KEX2 proteinase can specifically cleave large recombinant proteins, for example, a protein consisting of a gamma-interferon fragment linked to HIV1-proteinase via a Lys-Arg-containing peptide. Biochem J, 1997 Oct 15, 327 ( Pt 2), 545 - 52 Regulation of inositol lipid-specific phospholipase cdelta by changes in Ca2+ ion concentrations; Allen V et al.; Studies of inositol lipid-specific phospholipase C (PLC) have elucidated the main regulatory pathways for PLCbeta and PLCgamma but the regulation of PLCdelta isoenzymes still remains obscure . Here we demonstrate that an increase in Ca2+ ion concentration within the physiological range (0.1-10 microM) is sufficient to stimulate PLCdelta1, but not PLCgamma1 and PLCbeta1, to hydrolyse cellular inositol lipids present in permeabilized cells . The activity of PLCdelta1 is further enhanced in the presence of phosphatidylinositol transfer protein (PI-TP) . Both full activation by Ca2+ ions and stimulation in the presence of PI-TP require an intact PH domain involved in the membrane attachment of PLCdelta1 . The physiological implication of this study is that PLCdelta1 could correspond to a previously uncharacterized PLC responsible for Ca2+ ion-stimulated inositol lipid hydrolysis observed in many cellular systems. Spectrochim Acta A Mol Biomol Spectrosc, 1997 Sep, 53A(10), 1633 - 6 Study on the binding of cerium(III) ion and spermine to TRNA(Phe); Meng JX et al.; Fluorescence of Ce(III) aqua ion at pH 6.0 is found to be in good linear relationship with its concentration, and the intensity is strong enough to be employed to determine its concentration . TRNA(Phe) evidently quench this fluorescence . Fluorescence titration experiments were performed to Ce(III)-tRNA(Phe) system, the binding number and association constant was estimated with a Scatchard plot . Two classes of binding sites with association constant of 5.2 x 10(7) and 4.4 x 10(6) M, respectively, were found . Addition of spermine slightly decrease binding number and association constant of Ce(III) ion. Gene, 1997 Oct 15, 199(1-2), 181 - 94 Ku80 gene expression is Sp1-dependent and sensitive to CpG methylation within a novel cis element; Ludwig DL et al.; The Ku70/80 complex, known as Ku, constitutes the DNA end binding component of the DNA-dependent protein kinase (DNA-PK) . We have characterized the promoter region of the mouse and human Ku80 genes to delineate transcriptional elements necessary for basal gene expression and proliferation-dependent regulation . Consensus Sp1 recognition elements were identified in both promoters, and were determined to be essential for basal expression . We further identified a near-perfect palindrome of 21 base pairs located immediately 5' to one Sp1 element . This sequence was present once within the mouse Ku80 promoter and seven times, in a head-to-tail tandem array, within the human Ku80 promoter . This sequence possessed homology with a methylation-sensitive promoter element, Enh2, present in the LTR of mouse intractisternal A-particles . Promoter deletion studies and expression analysis of in-vitro methylated reporter gene constructs provided strong evidence that, in vivo, this repeat sequence regulates Ku80 gene expression in cis, through a mechanism involving CpG methylation . Evidence is also presented, suggesting that Ku is directly involved in this regulatory process. Gene, 1997 Oct 15, 199(1-2), 139 - 43 Drosophila DPP2C1, a novel member of the protein phosphatase 2C (PP2C) family; Dick T et al.; We report the molecular cloning, chromosome mapping and developmental transcription pattern of a putative serine/threonine protein phosphatase 2C (PP2C), DPP2C1, from Drosophila melanogaster . The 6-kb transcript of this first Drosophila PP2C gene encodes a 1428-aa deduced protein . The DPP2C1 protein contains a approximately 330-aa PP2C-like catalytic domain flanked by extensive N- and C-terminal sequences showing no similarities to other PP2Cs . The dpp2c1 gene maps to 4E1-2 on the X chromosome, 1.5 kb upstream of the ddlc1 gene . Northern blot analyses showed that dpp2c1 transcription is developmentally regulated, accumulating maximally during early (0-6 h) and late (12-24 h) embryogensis . The presented molecular characterisation provides the basis for a genetic dissection of DPP2C1 function. Proc Natl Acad Sci U S A, 1997 Nov 11, 94(23), 12580 - 5 The affected gene underlying the class K glycosylphosphatidylinositol (GPI) surface protein defect codes for the GPI transamidase; Yu J et al.; The final step in glycosylphosphatidylinositol (GPI) anchoring of cell surface proteins consists of a transamidation reaction in which preassembled GPI donors are substituted for C-terminal signal sequences in nascent polypeptides . In previous studies we described a human K562 cell mutant, termed class K, that accumulates fully assembled GPI units but is unable to transfer them to N-terminally processed proproteins . In further work we showed that, unlike wild-type microsomes, microsomes from these cells are unable to support C-terminal interaction of proproteins with the small nucleophiles hydrazine or hydroxylamine, and that the cells thus are defective in transamidation . In this study, using a modified recombinant vaccinia transient transfection system in conjunction with a composite cDNA prepared by 5' extension of an existing GenBank sequence, we found that the genetic element affected in these cells corresponds to the human homolog of yGPI8, a gene affected in a yeast mutant strain exhibiting similar accumulation of GPI donors without transfer . hGPI8 gives rise to mRNAs of 1.6 and 1.9 kb, both encoding a protein of 395 amino acids that varies in cells with their ability to couple GPIs to proteins . The gene spans approximately 25 kb of DNA on chromosome 1 . Reconstitution of class K cells with hGPI8 abolishes their accumulation of GPI precursors and restores C-terminal processing of GPI-anchored proteins . Also, hGPI8 restores the ability of microsomes from the mutant cells to yield an active carbonyl in the presence of a proprotein which is considered to be an intermediate in catalysis by a transamidase. Proc Natl Acad Sci U S A, 1997 Nov 11, 94(23), 12521 - 6 CDC45 is required in conjunction with CDC7/DBF4 to trigger the initiation of DNA replication; Owens JC et al.; The initiation of DNA replication in Saccharomyces cerevisiae requires the protein product of the CDC45 gene . We report that although Cdc45p is present at essentially constant levels throughout the cell cycle, it completes its initiation function in late G1, after START and prior to DNA synthesis . Shortly after mitosis, cells prepare for initiation by assembling prereplicative complexes at their replication origins . These complexes are then triggered at the onset of S phase to commence DNA replication . Cells defective for CDC45 are incapable of activating the complexes to initiate DNA replication . In addition, Cdc45p and Cdc7p/Dbf4p, a kinase implicated in the G1/S phase transition, are dependent on one another for function . These data indicate that CDC45 functions in late G1 phase in concert with CDC7/DBF4 to trigger initiation at replication origins after the assembly of the prereplicative complexes. Proc Natl Acad Sci U S A, 1997 Nov 11, 94(23), 12473 - 8 Cells that register logical relationships among proteins; Xu CW et al.; Two-hybrid methods have augmented the classical genetic techniques biologists use to assign function to genes . Here, we describe construction of a two-bait interaction trap that uses yeast cells to register more complex protein relationships than those detected in existing two-hybrid systems . We show that such cells can identify bridge or connecting proteins and peptide aptamers that discriminate between closely related allelic variants . The protein relationships detected by these cells are analogous to classical genetic relationships, but lend themselves to systematic application to the products of entire genomes and combinatorial libraries . We show that, by performing logical operations on the phenotypic outputs of these complex cells and existing two-hybrid cells, we can make inferences about the topology and order of protein interactions . Finally, we show that cells that register such relationships can perform logical operations on protein inputs . Thus these cells will be useful for analysis of gene and allele function, and may also define a path for construction of biological computational devices. Proc Natl Acad Sci U S A, 1997 Nov 11, 94(23), 12413 - 8 Dystrobrevin and dystrophin: an interaction through coiled-coil motifs; Sadoulet-Puccio HM et al.; Dystrobrevin, a dystrophin-related and -associated protein, has been proposed to be important in the formation and maintenance of the neuromuscular junction . Dystrobrevin coprecipitates with both the acetylcholine receptor complex as well as the dystrophin glycoprotein complex . Although the nature of dystrobrevin's association with the dystrophin glycoprotein complex remains unclear, it is known that dystrobrevin binds directly to the syntrophins, a heterologous group of dystrophin-associated proteins . Using the yeast two-hybrid system to identify protein-protein interactions, we present evidence for the heterodimerization of dystrobrevin directly with dystrophin . The C terminus of dystrobrevin binds specifically to the C terminus of dystrophin . We further refined this site of interaction to these proteins' homologous coiled-coil motifs that flank their respective syntrophin-binding sites . We also show that the interaction between the dystrobrevin and dystrophin coiled-coil domains is specific and is not due to a nonspecific coiled-coil domain interaction . From the accumulated evidence of protein-protein interactions presented here and elsewhere, we propose a partially revised model of the organization of the dystrophin-associated glycoprotein complex. Toxicol Appl Pharmacol, 1997 Nov, 147(1), 93 - 100 Additive estrogenic activities of a binary mixture of 2',4',6'-trichloro- and 2',3',4',5'-tetrachloro-4-biphenylol; Ramamoorthy K et al.; The estrogenic activity of 2',4',6'-trichloro-4-biphenylol (HO-PCB3), 2',3',4',5'-tetrachloro-4-biphenylol (HO-PCB4), and an equimolar mixture of both compounds (HO-PCB3/HO-PCB4) was investigated in the 21-day-old B6C3F1 mouse uterus, MCF-7 and MDA-MB-231 human breast cancer cells, HepG2 cells, and in a yeast-based reporter gene assay . Treatment of the animals with 17beta-estradiol (E2) (0.02 microg/kg/day x3) resulted in increased uterine wet weight, peroxidase activity and progesterone receptor binding . Treatment with 18, 73, 183 or 366 micromol/kg (x3) doses of HO-PCB3, HO-PCB4, or HO-PCB3/HO-PCB4 (equimolar) caused a dose-dependent increase in estrogenic activity; a maximal-induced response was not observed at any dose and the activity of the mixture was additive . Binding of E2, HO-PCB3, HO-PCB4, and HO-PCB3/HO-PCB4 to the mouse uterine estrogen receptor (ER) was determined in a competitive binding assay using {3H}E2 as the radioligand . The IC50 values were 1.1 x 10(-8), 3.4 x 10(-6), 9.9 x 10(-7), and 4.25 x 10(-6) m, respectively . HO-PCB3 and HO-PCB4 maximally induced MCF-7 cell proliferation, rat creatine kinase, and human complement C3 (C3-LUC) reporter gene activity at concentrations of 10(-5) to 10(-6) m, and these compounds were 10(3) to 10(4) less potent than E2 . The HO-PCB3/HO-PCB4 mixture was active at the high concentration (10(-5) m) and was additive for these responses . HO-PCB3 and HO-PCB4 also exhibited estrogenic activity in human HepG2 cells cotransfected with C3-LUC and an ER expression plasmid, and the estrogenic activity of the HO-PCB mixture was additive . Similar results were obtained in yeast transformed with the human ER and a double estrogen responsive element upstream of the beta-gal reporter gene . The effects of variable ER expression on the potential synergistic interactions of HO-PCB3/HO-PCB4 were investigated in HepG2 cells cotransfected with C3-LUC (405 ng/well) and variable amounts of ER expression plasmid (270, 27, 2.7, or 0.27 ng/well) . The results show that as ER levels decreased, the magnitude of the induction response by E2, HO-PCB3, HO-PCB4, and HO-PCB3/HO-PCB4 also decreased . However, the activities of the HO-PCB mixture were additive at high and low levels of ER . Similar results were obtained in MDA-MB-231 cells cotransfected with C3-LUC and variable amounts of ER expression plasmid . The results of this study demonstrate that for several estrogen-responsive assays in the mouse uterus; MCF-7, HepG2, and MDA-MBA-231 human cancer cells; and a yeast based-reporter gene assay, both HO-PCB3 and HO-PCB4 exhibited estrogenic activity . The estrogenic activity of an equimolar mixture of these compounds was additive at high and low levels of ER expression . Mol Plant Microbe Interact, 1997 Nov, 10(8), 984 - 93 Targeted disruption of a fungal G-protein beta subunit gene results in increased vegetative growth but reduced virulence; Kasahara S et al.; Targeted disruption of two G-protein alpha subunit genes in the chestnut blight fungus Cryphonectria parasitica revealed roles for the Gi alpha subunit CPG-1 in fungal reproduction, virulence, and vegetative growth . A second G alpha subunit, CPG-2, was found to be dispensable for these functions . We now report the cloning and targeted disruption of a C . parasitica G-protein beta subunit gene . The deduced amino acid sequence encoded by this gene, designated cpgb-1, was found to share 66.2, 65.9, and 66.7% amino acid identity with G beta homologues from human, Drosophila, and Dictyostelium origins, respectively, but only 39.7% identity with the Saccharomyces cerevisiae G beta homologue STE4 product . Low stringency Southern hybridization failed to detect any related G beta subunit genes in C . parasitica . Targeted disruption of cpgb-1 resulted in several of the changes previously reported to accompany disruption of the C . parasitica Gi alpha subunit gene cpg-1 . These included very significant reductions in pigmentation, asexual sporulation, and virulence . In contrast to results obtained for Gi alpha gene disruption, the reduction in virulence resulting from the disruption of a G beta gene was accompanied by increased, rather than decreased, vegetative growth on synthetic medium . The relevance of these results to mechanisms of fungal virulence is considered. Microbiology, 1997 Oct, 143 ( Pt 10), 3263 - 72 Dipeptidyl aminopeptidase processing and biosynthesis of alkaline extracellular protease from Yarrowia lipolytica; Matoba S et al.; Alkaline extracellular protease (AEP) from Yarrowia lipolytica is synthesized as a precursor with a 157 aa prepro-region . Signal peptide cleavage was shown to occur after Ala15 by N-terminal amino acid radiosequencing of the largest intracellular AEP precursor . AEP proteolytic activity was not required for AEP processing . After a change of the putative active site Ser to Ala, inactive AEP with the same mobility on SDS-PAGE as wild-type mature AEP was secreted . The role of dipeptidyl aminopeptidase (DPAPase) activity in AEP processing was also investigated . Mutations early in the -X-Ala- and -X-Pro- dipeptide stretch (Pro17 to Met which should prevent DPAPase processing and Ala19 to Val which should allow removal of only the first dipeptide) did not prevent synthesis of active mature AEP nor did use of the DPAPase inhibitor ProboroPro . Deletion of the entire dipeptide stretch (Ala16 to Pro33) resulted in intracellular accumulation of an AEP precursor, which surprisingly was not glycosylated, and little or no secretion of AEP-related polypeptides . Expression of AEP in wild-type and dpp1 dap2 Saccharomyces cerevisiae strains (lacking both the Golgi and vacuolar DPAPases) resulted in secretion of only mature AEP and no AEP precursors . Transit times and levels of AEP secretion were similar for both strains . These results indicate that the KEX2-like cleavage after Lys156-Arg157, which yields mature active AEP can occur in the absence of DPAPase processing and that DPAPase processing is not necessary for secretion of mature active AEP. J Biol Chem, 1997 Nov 7, 272(45), 28695 - 703 Cloning and characterization of a human STE20-like protein kinase with unusual cofactor requirements; Schinkmann K et al.; We cloned and characterized a novel human member of the STE20 serine/threonine protein kinase family named mst-3 . Based on its domain structure, mst-3 belongs to the SPS1 subgroup of STE20-like proteins, which includes germinal center (GC) kinase, hematopoietic progenitor kinase (HPK), kinase homologous to STE20/SPS-1 (KHS), kinases responsive to stress (KRS1/2), the mammalian STE20-like kinases (mst1/2), and the recently published STE20/oxidant stress response kinase SOK-1 . mst-3 is most closely related to SOK-1, with 88% amino acid similarity in the kinase domain . The similarity of the mst-3 kinase domain to STE20 is 42% . The mst-3 transcript is ubiquitously expressed, and the protein was found in all human, mouse, and monkey cell lines tested . An in vitro kinase assay showed that mst-3 can phosphorylate basic exogenous substrates as well as itself . Interestingly, mst-3 prefers Mn2+ to Mg2+ as a divalent cation and can use both GTP and ATP as phosphate donors . Like SOK-1, mst-3 is activated by autophosphorylation . However, a physiological stimulus of mst-3 activity was not identified . mst-3 activity does not change upon exposure to several mitogenic and stress stimuli . Overexpression of mst-3 wild-type or kinase dead protein affects neither the extracellular signal-regulated kinases (ERK1/2 or ERK6), c-Jun N-terminal kinase (JNK), p38, nor pp70S6 kinase, suggesting that mst-3 is part of a novel signaling pathway. J Biol Chem, 1997 Nov 7, 272(45), 28501 - 11 Cell cycle-regulated expression, phosphorylation, and degradation of p55Cdc . A mammalian homolog of CDC20/Fizzy/slp1; Weinstein J; p55Cdc is a mammalian protein that shows high homology to the cell cycle proteins Cdc20p of Saccharomyces cerevisiae and the product of the Drosophila fizzy (fzy) gene, both of which contain WD repeats and are thought to be required for the metaphase-anaphase transition . The fzy mutants exhibit a metaphase arrest phenotype, which is accompanied by stabilization of cyclins A and B, leading to the hypothesis that fzy function is required for cell cycle-regulated ubiquitin-mediated proteolysis . p55Cdc expression was initiated at the G1/S transition and steady state levels of p55Cdc were highest at M and lowest in G1 . Inhibition of the 26 S proteasome prevented both mitotic exit and loss of p55Cdc at the M/G1 transition, suggesting that p55Cdc degradation was mediated by the cell cycle-regulated proteolytic pathway . Immune complexes of p55Cdc obtained at different cell cycle stages showed a variety of proteins with dramatic differences observed in the pattern of associated proteins during the transition from G2 to M . Immunolocalization of p55Cdc demonstrated dynamic changes in p55Cdc localization as the cells transit mitosis . p55Cdc appears to act as a regulatory protein interacting with several other proteins, perhaps via its seven WD repeats, at multiple points in the cell cycle. J Biol Chem, 1997 Nov 7, 272(45), 28407 - 14 HRX leukemic fusion proteins form a heterocomplex with the leukemia-associated protein SET and protein phosphatase 2A; Adler HT et al.; One of the most common chromosomal abnormalities in acute leukemia is a reciprocal translocation involving the HRX gene at chromosome locus 11q23, resulting in HRX fusion proteins . Using the yeast two-hybrid system, in vitro binding studies, and human cell culture coimmunoprecipitation experiments, we show here that a region of the HRX protein that is consistently retained in HRX leukemic fusion proteins interacts directly with SET, another protein implicated in leukemia . We have identified the binding sites on HRX for SET and show that these sequences are clustered near the A.T hooks that have been shown to bind DNA . We also show that carboxyl-terminal SET sequences, possibly the acidic tail of SET, bind to HRX . We have also found serine/threonine-specific protein phosphatase activity in anti-HRX coimmunoprecipitates . Using the phosphatase inhibitor okadaic acid and Western blotting, the phosphatase was identified as protein phosphatase 2A (PP2A) . Mutation of a single amino acid in one of the SET binding sites of HRX resulted in lower amounts of both coimmunoprecipitated SET protein and coimmunoprecipitated PP2A . These results suggest that the leukemogenic effects of HRX fusion proteins may be related to interactions with SET and PP2A. J Biol Chem, 1997 Nov 7, 272(45), 28247 - 51 Identification of HsORC4, a member of the human origin of replication recognition complex; Quintana DG et al.; A new member of human origin recognition complex (ORC) has been cloned and identified as the human homologue of Saccharomyces cerevisiae ORC4 . HsORC4 is a 45-kDa protein encoded by a 2.2-kilobase mRNA whose amino acid sequence is 29% identical to ScORC4 . HsORC4 has a putative nucleotide triphosphate binding motif that is not seen in ScORC4 . HsORC4P also reveals an unsuspected homology to the ORC1-Cdc18 family of proteins . HsORC4 mRNA expression and protein levels remain constant through the cell cycle . HsORC4P is coimmunoprecipitated from cell extracts with another subunit of human ORC, HsORC2P, consistent with it being a part of the putative human origin recognition complex. Curr Opin Biotechnol, 1997 Oct, 8(5), 629 - 34 Gene expression systems in the development of high-throughput screens; Jayawickreme CK et al.; Recent advances in the development of combinatorial automated chemical synthesis, robotic sample handling, and data collection and analysis have significantly increased the number of compounds available for screening against potential therapeutic targets . The implementation of highly sensitive in vitro biochemical and cell-based high-throughput screening assays is essential to facilitate the rapid identification of selective and potent lead molecules from compound libraries . The ability to easily produce functional proteins in sufficient quantities for in vitro biochemical assays and to devise useful cell-based systems is dependent on the successful application of a variety of gene expression systems. Nature, 1997 Oct 30, 389(6654), 974 - 8 The cerebellar leucine-rich acidic nuclear protein interacts with ataxin-1; Matilla A et al.; Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder characterized by ataxia, progressive motor deterioration, and loss of cerebellar Purkinje cells . SCA1 belongs to a growing group of neurodegenerative disorders caused by expansion of CAG repeats, which encode glutamine . Although the proteins containing these repeats are widely expressed, the neurodegeneration in SCA1 and other polyglutamine diseases selectively involves a few neuronal subtypes . The mechanism(s) underlying this neuronal specificity is unknown . Here we show that the cerebellar leucine-rich acidic nuclear protein (LANP) interacts with ataxin-1, the SCA1 gene product . LANP is expressed predominantly in Purkinje cells, the primary site of pathology in SCA1 . The interaction between LANP and ataxin-1 is significantly stronger when the number of glutamines is increased . Immunofluorescence studies demonstrate that both LANP and ataxin-1 colocalize in nuclear matrix-associated subnuclear structures . The features of the interaction between ataxin-1 and LANP, their spatial and temporal patterns of expression, and the colocalization studies indicate that cerebellar LANP is involved in the pathogenesis of SCA1. Pharmacogenetics, 1997 Oct, 7(5), 381 - 90 Role of CYP2D6 in the N-hydroxylation of procainamide; Lessard E et al.; Sequential oxidations at the arylamine moiety of the procainamide molecule leading to the formation of N-hydroxyprocainamide and its nitroso derivative may be responsible for lupus erythematosus observed in patients treated with the drug . The objective of the present study was to characterize major cytochrome P450 isozyme(s) involved in the N-hydroxylation of procainamide . Firstly, incubations were performed with microsomes from either lymphoblastoid cells or yeast transfected with cDNA encoding for specific human cytochrome P450 isozymes . Experiments performed with these enzyme expression systems indicated that the highest formation rate of N-hydroxyprocainamide was observed in the presence of CYP2D6 enriched microsomes . Additional experiments demonstrated that the formation rate of N-hydroxyprocainamide by CYP2D6 enriched microsomes was decreased from 45 +/- 4% to 93 +/- 1% by quinidine at concentrations ranging from 30 nM to 100 microM (all p < 0.05 vs control) and by approximately 75% by antibodies directed against CYP2D6 . Secondly, incubations were performed with microsomes prepared from 15 human liver samples . Using this approach, an excellent correlation was observed between the formation rate of N-hydroxyprocainamide and dextromethorphan O-demethylase activity (CYP2D6; r = 0.9305; p < 0.0001) . In contrast, no correlation could be established between N-hydroxyprocainamide formation rate and caffeine N3-demethylase (CYP1A2), coumarin 7-hydroxylase (CYP2A6), S-mephenytoin N-demethylase (CYP2B6), tolbutamide methlhydroxylase (CYP2C9), S-mephenytoin 4'-hydroxylase (CYP2C19), chlorzoxazone 6-hydroxylase (CYP2E1), dextromethorphan N-demethylase (CYP3A4), testosterone 6 beta-hydroxylase (CYP3A4/5) or lauric acid 12-hydroxylase (CYP4A11) activities . Furthermore, formation rate of N-hydroxyprocainamide was decreased in a concentration-dependent manner by quinidine (300 nM to 100 microM) and by antibodies directed against CYP2D6 but not by furafylline 20 microM (CYP1A2), ketoconazole 1 microM (CYP3A4), sulfaphenazole 10 microM (CYP2C9) or antibodies directed against CYP1A1/1A2, CYP2C, CYP2A6, CYP2E1 or CYP3A4/3A5 . In conclusion, the results obtained in the present study demonstrate that CYP2D6 is the major human cytochrome P450 isozyme involved in the formation of the reactive metabolite of procainamide, namely N-hydroxyprocainamide. Mech Ageing Dev, 1997 Dec, 98(3), 223 - 30 Molecular genetic approaches to the genes of longevity, aging and neurodegeneration in mammals; Mori N; Accumulative evidence suggests that species life-span is determined, at least in part, genetically . Recent cloning works using yeast (Saccharomyces cerevisiae) and worms (Caenorhabditis elegans) revealed several potential candidate genes, e.g . SIR4, a transcriptional silencing factor, and age-1, a putative signal transduction molecule, respectively, that may be involved in determining and/or regulating species life-span in lower organisms . It is, however, not clear yet whether mammalian homologs of these genes are also relevant to controlling longevity in higher organisms . In mice and humans several silencing factors are essential for cell-type specific gene expression . A variety of signal transducing molecules are also known to play important roles in mammals . I will briefly summarize recent progress in molecular genetic studies on such longevity-related genes, and discuss these results with our recent findings on a neural-selective silencing factor and a neural-specific signaling molecule that are important for functioning of the nervous system. Int Immunol, 1997 Oct, 9(10), 1607 - 13 Molecular genetic characterization of XRCC4 function; Mizuta R et al.; XRCC4 is a generally expressed protein of 334 amino acids that is involved in the repair of DNA double-strand breaks and in V(D)J recombination, but its function is unknown . In this study, we have used a mutational approach and the yeast two-hybrid method to perform an initial characterization of this protein . We show that the XRCC4 protein is located in the nucleus . We also demonstrate that several potential phosphorylation sites are not required for XRCC4 function in a transient V(D)J recombination assay . In addition, we show that XRCC4 forms a homodimer in vivo with the homodimerization domain being located within amino acids 115-204 . Finally, we define a core domain of XRCC4 that functions in V(D)J recombination and comprises amino acids 18-204 . Potential functions of XRCC4 are discussed. Curr Top Dev Biol, 1998, 37, 201 - 39 Caught in the act: deducing meiotic function from protein immunolocalization; Ashley T et al.; Meiotic division comprises a complex series of events, many of which are unique in the life cycle of the organism . The process utilizes both proteins that participate in normal mitotic cell cycle progression and DNA damage repair and proteins expressed only during meiosis . Until recently, few meiotic protein participants had been identified and characterized, but several recent developments have changed this situation . Proteins can be selected for study based on their cDNA sequence and similarity to known proteins with "suspicious" repair/recombination or cell cycle activity and antibodies against these proteins applied to meiotic nuclei to test for activity . With the development of gene sequence data bases from many organisms, similarity to a known protein need not be based on the same or even a closely related species . Potential interactions between two or more proteins can be identified and involvement in a common process inferred based on antibody colocalization . The gene sequence can be disrupted and the effect on meiotic progression directly examined . Previously identified structures, the synaptonemal complex (SC) and both early and late recombination nodules (RNs), provide structural and temporal landmarks that assist in inferring meiotic activity of the protein being studied . Mammalian meiosis is especially attractive for these kinds of studies since spermatocyte and oocyte nuclei are large with distinct nuclear organelles and since meiosis is highly protracted, occurring over a period of several days . In this chapter, an approach to the study of mammalian meiosis based on use of specific antibodies is outlined and methods of coupling this approach to other techniques, such as targeted gene disruption or chromosome aberrations, are described . Some of the proteins already identified as participants in meiotic prophase are reviewed and their presumed functions discussed. Curr Top Dev Biol, 1998, 37, 117 - 40 Functions of DNA repair genes during meiosis; Cummings WJ et al.; One of the most basic functions in any organism is DNA repair . In addition, programmed DNA "damage," in the form of DNA double-strand breaks (DSBs), is a regular part of the physiology of most organisms . There are three main types of DSB repair: homologous recombination; single-strand annealing; and nonhomologous end joining . The gene products known to be required for these repair processes are conserved in evolution, but the relative dependence on different pathways for DSB repair is different when systems are compared . In the yeast Saccharomyces cerevisiae, the formation and repair of DNA double-strand breaks (DSBs) is apparently an essential feature of meiotic recombination . However, it is not clear whether DSBs are a conserved feature of meiotic recombination in eukaryotes . The basidiomycete Coprinus cinereus presents an experimental system which is amenable to genetic analysis, processes DSBs in a manner similar to complex eukaryotes, and has a naturally synchronous meiosis . An understanding of the functions of conserved genes in DSB repair in C . cinereus and other similar systems will help to determine whether DSB repair is a unifying theme in meiotic recombination or whether conserved gene products have other essential functions that tie together DNA repair and meiosis. EMBO J, 1997 Nov 3, 16(21), 6521 - 34 Dual role for fimbriata in regulating floral homeotic genes and cell division in Antirrhinum; Ingram GC et al.; The fimbriata (fim) gene of Antirrhinum affects both the identity and arrangement of organs within the flower, and encodes a protein with an F-box motif . We show that FIM associates with a family of proteins, termed FAPs (FIM-associated proteins), that are closely related to human and yeast Skp1 proteins . These proteins form complexes with F-box-containing partners to promote protein degradation and cell cycle progression . The fap genes are expressed in inflorescence and floral meristems in a pattern that incorporates the domain of fim expression, supporting an in vivo role for a FIM-FAP complex . Analysis of a series of novel fim alleles shows that fim plays a key role in the activation of organ identity genes . In addition, fim acts in the regions between floral organs to specify the correct positioning and maintenance of morphological boundaries . Taking these results together, we propose that FIM-FAP complexes affect both gene expression and cell division, perhaps by promoting selective degradation of regulatory proteins . This may provide a mechanism by which morphological boundaries can be aligned with domains of gene expression during floral development. EMBO J, 1997 Nov 3, 16(21), 6374 - 83 Identification of a species-specific inhibitor of glycosylphosphatidylinositol synthesis; Sutterlin C et al.; Glycosylphosphatidylinositol (GPI)-anchoring represents a mechanism for attaching proteins to the cell surface that is used among all eukaryotes . A common core structure, EthN-P-Man3-GlcN-PI, is synthesized by sequential transfer of sugars and ethanolamine-P to PI and is highly conserved between organisms . We have screened for natural compounds that inhibit GPI-anchoring in yeast and have identified a terpenoid lactone, YW3548, that specifically blocks the addition of the third mannose to the intermediate structure Man2-GlcN-acyIPI . Consistent with the block in GPI synthesis, YW3548 prevents the incorporation of {3H}myo-inositol into proteins, transport of GPI-anchored proteins to the Golgi and is toxic . The compound inhibits the same step of GPI synthesis in mammalian cells, but has no significant activity in protozoa . These results suggest that despite the conserved core structure, the GPI biosynthetic machinery may be different enough between mammalian and protozoa to represent a target for anti-protozoan chemotherapy. EMBO J, 1997 Nov 3, 16(21), 6346 - 54 The large subunit of replication factor C is a substrate for caspase-3 in vitro and is cleaved by a caspase-3-like protease during Fas-mediated apoptosis; Rheaume E et al.; Caspase-3 is an ICE-like protease activated during apoptosis induced by different stimuli . Poly(ADP-ribose) polymerase (PARP), the first characterized substrate of caspase-3, shares a region of homology with the large subunit of Replication Factor C (RF-C), a five-subunit complex that is part of the processive eukaryotic DNA polymerase holoenzymes . Caspase-3 cleaves PARP at a DEVD-G motif present in the 140 kDa subunit of RF-C (RFC140) and evolutionarily conserved . We show that cleavage of RFC140 during Fas-mediated apoptosis in Jurkat cells and lymphocytes results in generation of multiple fragments . Cleavage is inhibited by the caspase-3-like protease inhibitor Ac-DEVD-CHO but not the caspase-1/ICE-type protease inhibitor Ac-YVAD-CHO . In addition, recombinant caspase-3 cleaves RFC140 in vitro at least at three different sites in the C-terminal half of the protein . Using amino-terminal microsequencing of radioactive fragments, we identified three sites: DEVD723G, DLVD922S and IETD1117A . We did not detect cleavage of small subunits of RF-C of 36, 37, 38 and 40 kDa by recombinant caspase-3 or by apoptotic Jurkat cell lysates . Cleavage of RFC140 during apoptosis inactivates its function in DNA replication and generates truncated forms that further inhibit DNA replication . These results identify RFC140 as a critical target for caspase-3-like proteases and suggest that caspases could mediate cell cycle arrest. Br J Pharmacol, 1997 Oct, 122(3), 493 - 503 Role of peroxynitrite and activation of poly (ADP-ribose) synthase in the vascular failure induced by zymosan-activated plasma; Cuzzocrea S et al.; 1 . Zymosan is a wall component of the yeast Saccharomyces Cerevisiae . Injection of zymosan into experimental animals is known to produce an intense inflammatory response . Recent studies demonstrated that the zymosan-induced inflammatory response in vivo can be ameliorated by inhibitors of nitric oxide (NO) biosynthesis . The cytotoxic effects of NO are, in part, mediated by the oxidant preoxynitrite and subsequent activation of the nuclear enzyme poly (ADP-ribose) synthetase (PARS) . In the present in vitro study, we have investigated the cellular mechanisms of vascular failure elicited by zymosan-activated plasma and the contribution of peroxynitrite production and activation of PARS to the changes . 2 . Incubation of rat aortic smooth muscle cells with zymosan-activated plasma (ZAP) induced the production of nitrite, the breakdown product of NO, due to the expression of the inducible isoform of NO synthase (iNOS) over 6 24 h . In addition, ZAP triggered the production of peroxynitrite in these cells, as measured by the oxidation of the fluorescent dye dihydrorhodamine 123 and by nitrotyrosine Western blotting . 3 . Incubation of the smooth muscle cells with ZAP induced DNA single strand breakage and PARS activation . These effects were reduced by inhibition of NOS with NG-methyl-L-arginine (L-NMA, 3 mM), and by glutathione (3 mM), a scavenger of peroxynitrite . The PARS inhibitor 3-aminobenzamide (1 mM) inhibited the ZAP-induced activation of PARS . 4 . Incubation of thoracic aortae with ZAP in vitro caused a reduction of the contractions of the blood vessels to noradrenaline (vascular hyporeactivity) and elicited a reduced responsiveness to the endothelium-dependent vasodilator acetylcholine (endothelial dysfunction) . 5 . Preincubation of the thoracic aortae with L-NMA (1 mM), glutathione (3 mM) or by the PARS inhibitor 3-aminobenzamide (1 mM) prevented the development of vascular hyporeactivity in response to ZAP . Moreover, glutathione and 3-aminobenzamide treatment protected against the ZAP-induced development of endothelial dysfunction . The PARS-related loss of the vascular contractility was evident at 30 min after incubation in endothelium-intact, but not in endothelium-denuded vessels and also manifested at 6 h after incubation with ZAP in endothelium-denuded rings . The acute response is probably related, therefore, to peroxynitrite formation (involving the endothelial NO synthase), whereas the delayed response may be related to the expression of iNOS in the smooth muscle . 6 . The data obtained suggest that zymosan-activated plasma causes vascular dysfunction by inducing the simultaneous formation of superoxide and NO . These radicals combine to form peroxynitrite, which, in turn causes DNA injury and PARS activation . The protective effect of 3-aminobenzamide demonstrates that PARS activation contributes both to the development of vascular hyporeactivity and endothelial dysfunction during the vascular failure induced by ZAP. J Biol Chem, 1997 Oct 31, 272(44), 27582 - 8 FKBP12 binds the inositol 1,4,5-trisphosphate receptor at leucine-proline (1400-1401) and anchors calcineurin to this FK506-like domain; Cameron AM et al.; The immunophilin FKBP12 is one of the most abundant and conserved proteins in biology . It is the primary receptor for the immunosuppressant actions of the drug FK506 in whose presence FKBP12 binds to and inhibits calcineurin, disrupting interleukin formation in lymphocytes . The physiologic functions of FKBP12 are less clear, although the protein has been demonstrated to physiologically interact with the inositol 1,4,5-trisphosphate receptor (IP3R), the ryanodine receptor, and the type 1 transforming growth factor beta receptor . We now report that FKBP12 binds the IP3R at residues 1400-1401, a leucyl-prolyl dipeptide epitope that structurally resembles FK506 . We further demonstrate that binding to IP3R at this site enables FKBP12 to interact with calcineurin, presumably to anchor the phosphatase to IP3R and modulate the receptor's phosphorylation status . We propose that FK506 promotes an FKBP12-calcineurin interaction by mimicking structurally similar dipeptide epitopes present within proteins that use FKBP12 to anchor calcineurin to the appropriate physiologic substrates. Biochemistry, 1997 Oct 14, 36(41), 12554 - 9 Structural and functional analysis of peptidyl oligosaccharyl transferase inhibitors; Kellenberger C et al.; The peptide cyclo(hex-Amb(1)-Cys(2))-Thr(3)-Val(4)-Thr(5)-Nph(6)-NH2 was previously shown to be a slow, tight-binding inhibitor (Ki = 37 nM) of the yeast oligosaccharyl transferase (OT) {Hendrickson et al . (1996) J . Am . Chem . Soc . 118, 7636-7637} . This enzyme catalyzes the transfer of a carbohydrate moiety to an asparagine residue in the consensus sequence Asn-Xaa-Thr/Ser . Herein we present a study of the contribution of the residues in positions 1, 3, 4, and 5 to OT binding . Replacement of the threonine (residue 3) by valine or (S)-2-aminobutyric acid dramatically reduced the potency of the inhibitor while, surprisingly, the incorporation of an additional methylene into the side chain of residue 1 {(S)-2,3-diaminobutyric acid changed to ornithine} had very little effect . Variants with acidic, basic, hydrophilic/polar, and hydrophobic side chains in positions 4 and 5 were also evaluated for both yeast and porcine liver OT inhibition . This aspect of the study reveals that basic (lysine) and acidic (glutamic acid) residues are detrimental to the binding, whereas hydrophobic (valine) and polar/hydrophilic (threonine) residues are both well tolerated . The kinetic behavior of substrate analogs {cyclo(hex-Asn(1)-Cys(2))-Thr(3)-Xaa(4)-Yaa(5)-Nph-NH2} corresponding to inhibitors of weak, medium, and strong potency was also examined in order to provide insight into the nature of these inhibitors. J Biol Chem, 1997 Oct 31, 272(44), 28102 - 6 The Mr 18,000 subunit of the peripheral-type benzodiazepine receptor exhibits both benzodiazepine and isoquinoline carboxamide binding sites in the absence of the voltage-dependent anion channel or of the adenine nucleotide carrier; Joseph-Liauzun E et al.; The peripheral type benzodiazepine receptor (PBR) binds benzodiazepines such as RO5-4864 and isoquinoline carboxamide derivatives such as PK11195 . This receptor includes an Mr 18,000 isoquinoline-binding subunit predominantly located in mitochondrial mem- branes . This protein has been found to copurify with two other mitochondrial proteins, namely the outer membrane voltage-dependent anion channel (VDAC), also known as mitochondrial porin, and the inner membrane adenine nucleotide carrier . In vitro reconstitution experiments suggested that the PBR was a multimeric complex in which the isoquinoline binding site was on the Mr 18,000 subunit, denoted pk18, whereas the benzodiazepine binding site required the association of this subunit with VDAC to be expressed . Untransformed cells of the yeast Saccharomyces cerevisiae are devoid of specific binding sites for isoquinolines and benzodiazepines, whereas yeast cells transformed with a pk18-expressing vector exhibit RO5-4864 and PK11195 binding sites that are pharmacologically identical to those of the PBR . To clarify the role of VDAC and of the adenine nucleotide carrier, if any, in the constitution of the benzodiazepine binding site, yeast host strains were constructed in which the corresponding genes had been knocked out . Mitochondria prepared from pk18-producing cells devoid of either VDAC or adenine nucleotide carrier exhibit both benzodiazepine and isoquinoline carboxamide binding sites with little or no change in the Kd values as compared with the wild-type background . These results rule out the contention that VDAC is indispensable for establishing the benzodiazepine binding site and are in agreement with the hypothesis that the Mr 18,000 subunit carries both the isoquinoline carboxamide and benzodiazepine binding domains. Cell, 1997 Oct 17, 91(2), 209 - 19 F-box proteins are receptors that recruit phosphorylated substrates to the SCF ubiquitin-ligase complex; Skowyra D et al.; We have reconstituted the ubiquitination pathway for the Cdk inhibitor Sic1 using recombinant proteins . Skp1, Cdc53, and the F-box protein Cdc4 form a complex, SCFCdc4, which functions as a Sic1 ubiquitin-ligase (E3) in combination with the ubiquitin conjugating enzyme (E2) Cdc34 and E1 . Cdc4 assembled with Skp1 functions as the receptor that selectively binds phosphorylated Sic1 . Grr1, an F-box protein involved in Cln destruction, forms complexes with Skp1 and Cdc53 and binds phosphorylated Cln1 and Cln2, but not Sic1 . Because the constituents of the SCF complex are members of protein families, SCFCdc4 is likely to serve as the prototype for a large class of E3s formed by combinatorial interactions of related family members . SCF complexes couple protein kinase signaling pathways to the control of protein abundance. Virology, 1997 Oct 13, 237(1), 33 - 45 HsN3 proteasomal subunit as a target for human immunodeficiency virus type 1 Nef protein; Rossi F et al.; HIV-1 Nef protein is important for pathogenicity, but its biochemical function remains obscure . To clarify its role, a yeast two-hybrid system (ths) screening was utilized to identify Nef cellular partners . Of 79 yeast clones harboring cDNAs for putative Nef binding proteins, 27 (34%) contained the coding region for HsN3 proteasomal subunit . HsN3 behaved as bona fide Nef partner in ths control crosses . Nef-HsN3 interaction was confirmed by in vitro binding experiments . In particular, recombinant Nef was able to capture the HsN3 subunit from a natural proteasome preparation . In Nef, the interacting region was mapped within aa 34-143, which span the structured portion of the protein, including the SH3-binding domain . In HsN3, Nef-binding portion was restricted to aa 73-249, and the tract 219-249-reminiscent of SH3 domain N-terminal 3/5ths-was shown to be essential, though not sufficient . Attempts to purify a Nef-HsN3 complex from transfected COS7 cells were unsuccessful . However, Nef was found to markedly downregulate intracellular levels of both a coexpressed HsN3 and the endogenous simian homologue . These results suggest that Nef, by binding to a subunit, might alter proteasome function in infected cells . Biochem Biophys Res Commun, 1997 Oct 20, 239(2), 439 - 46 Cloning and characterization of novel CIS family genes; Masuhara M et al.; We have reported two JAK-signaling modulators, CIS (cytokine-inducible SH2 protein) and JAB (JAK2 binding protein), which are structurally related . Here we cloned three additional CIS family genes (CIS2, CIS3, and CIS4) on the basis of an expression sequence tag (EST) database search . We also found at least two additional candidates of this gene family in the database . These genes were induced by erythropoietin and granulocyte-macrophage colony stimulating factor in certain hematopoietic cell lines . The SH2 domain and a C-terminal 40 amino acid region, designated the CIS homology domain (CH domain), are highly conserved in this family, while the N-terminal regions of these proteins share little similarity . A yeast two-hybrid assay and in vitro and in vivo binding assays revealed that in addition to JAB, CIS3 bound to the JAK2 tyrosine kinase domain (JH1), although the interaction of CIS3 with the JAK2-JH1 domain was much weaker than that of JAB . Transient expression of JAB and CIS3, but not other CISs, strongly inhibited leukemia inhibitory factor (LIF)-induced STAT3-reporter gene activation in 293 cells . Furthermore, constitutive overexpression of JAB and CIS3 in M1 leukemia cells prevented LIF-induced differentiation and growth arrest . Although the physiological function remains to be investigated, CIS family genes could play a role in the negative regulation of cytokine signaling by interacting with specific targets . Hum Mol Genet, 1997 Nov, 6(12), 2011 - 9 Isolation of a candidate human telomerase catalytic subunit gene, which reveals complex splicing patterns in different cell types; Kilian A et al.; Telomerase is a multicomponent reverse transcriptase enzyme that adds DNA repeats to the ends of chromosomes using its RNA component as a template for synthesis . Telomerase activity is detected in the germline as well as the majority of tumors and immortal cell lines, and at low levels in several types of normal cells . We have cloned a human gene homologous to a protein from Saccharomyces cerevisiae and Euplotes aediculatus that has reverse transcriptase motifs and is thought to be the catalytic subunit of telomerase in those species . This gene is present in the human genome as a single copy sequence with a dominant transcript of approximately 4 kb in a human colon cancer cell line, LIM1215 . The cDNA sequence was determined using clones from a LIM1215 cDNA library and by RT-PCR, cRACE and 3'RACE on mRNA from the same source . We show that the gene is expressed in several normal tissues, telomerase-positive post-crisis (immortal) cell lines and various tumors but is not expressed in the majority of normal tissues analyzed, pre-crisis (non-immortal) cells and telomerase-negative immortal (ALT) cell lines . Multiple products were identified by RT-PCR using primers within the reverse transcriptase domain . Sequencing of these products suggests that they arise by alternative splicing . Strikingly, various tumors, cell lines and even normal tissues (colonic crypt and testis) showed considerable differences in the splicing patterns . Alternative splicing of the telomerase catalytic subunit transcript may be important for the regulation of telomerase activity and may give rise to proteins with different biochemical functions. Carcinogenesis, 1997 Sep, 18(9), 1793 - 8 Metabolism of 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine (PhIP) by human cytochrome P4501B1; Crofts FG et al.; Cytochrome P4501B1 (CYP1B1) is the most recently identified member of the dioxin-inducible CYP1 family . CYP1B1 is constitutively expressed in most human tissues, including colon and breast, and can activate numerous chemically diverse carcinogens . We evaluated the metabolism of the dietary heterocyclic amine carcinogen 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine (PhIP) by microsomes from yeast expressing the human CYP1B1 protein . PhIP metabolites were analysed by HPLC with fluorescence and absorbance detection . We found that human CYP1B1 metabolizes PhIP to three products: N2-OH-PhIP, a mutagenic activation product; 4'-OH-PhIP, a detoxification product; and 2-OH-PhIP, the mutagenic potential of which is unknown . Metabolite identity was confirmed by co-elution with authentic standards and synchronous fluorescence spectroscopy . The identity of the 2-OH-PhIP standard was additionally confirmed by mass spectrometry . Kinetic studies of the formation of N2-OH-PhIP, 4'-OH-PhIP and 2-OH-PhIP by CYP1B1 indicated apparent Km values of 5.7 +/- 1.3, 2.2 +/- 0.5 and 1.3 +/- 0.2 microM, respectively . Apparent turnover rates were 0.40 +/- 0.03, 0.93 +/- 0.02 and 0.04 +/- 0.00 nmol product/min nmol P450, respectively . At saturating levels of substrate, CYP1B1-mediated formation of the non-mutagenic metabolite 4'-OH-PhIP was favored two-fold over that of the mutagenic metabolite, N2-OH-PhIP and >10-fold over that of 2-OH-PhIP . The formation of N2-OH-PhIP, a potent mutagen implicated in the etiology of human colon and breast cancer, indicates that CYP1B1 may play an important role in PhIP-mediated carcinogenesis. Nat Genet, 1997 Oct, 17(2), 215 - 7 Aconitase and mitochondrial iron-sulphur protein deficiency in Friedreich ataxia; Rotig A et al.; Friedreich ataxia (FRDA) is a common autosomal recessive degenerative disease (1/50,000 live births) characterized by a progressive-gait and limb ataxia with lack of tendon reflexes in the legs, dysarthria and pyramidal weakness of the inferior limbs . Hypertrophic cardiomyopathy is observed in most FRDA patients . The gene associated with the disease has been mapped to chromosome 9q13 (ref . 3) and encodes a 210-amino-acid protein, frataxin . FRDA is caused primarily by a GAA repeat expansion within the first intron of the frataxin gene, which accounts for 98% of mutant alleles . The function of the protein is unknown, but an increased iron content has been reported in hearts of FRDA patients and in mitochondria of yeast strains carrying a deleted frataxin gene counterpart (YFH1), suggesting that frataxin plays a major role in regulating mitochondrial iron transport . Here, we report a deficient activity of the iron-sulphur (Fe-S) cluster-containing subunits of mitochondrial respiratory complexes I, II and III in the endomyocardial biopsy of two unrelated FRDA patients . Aconitase, an iron-sulphur protein involved in iron homeostasis, was found to be deficient as well . Moreover, disruption of the YFH1 gene resulted in multiple Fe-S-dependent enzyme deficiencies in yeast . The deficiency of Fe-S-dependent enzyme activities in both FRDA patients and yeast should be related to mitochondrial iron accumulation, especially as Fe-S proteins are remarkably sensitive to free radicals . Mutated frataxin triggers aconitase and mitochondrial Fe-S respiratory enzyme deficiency in FRDA, which should therefore be regarded as a mitochondrial disorder. RNA, 1997 Oct, 3(10), 1153 - 8 In vivo HIV-1 frameshifting efficiency is directly related to the stability of the stem-loop stimulatory signal; Bidou L et al.; In many retroviruses, the expression of reverse transcriptase, protease, and integrase is dependent upon a -1 frameshift event . The frameshift signal is composed of a slippery sequence where the ribosome shifts, and a downstream stimulatory sequence . In most cases, the stimulatory sequence is a pseudoknot, but in some viruses, such as human immunodeficiency virus type 1 (HIV-1), a single stem-loop is involved . Here, we analyzed the precise role of the stem-loop thermodynamic stability . We tested the frameshifting stimulatory activity of a series of HIV-1-derived sequences showing a stepwise increment of the estimated deltaG degrees . These sequences were introduced at the junction of a lacZ-luc fusion gene cloned on a versatile expression vector, and the different constructs were tested in Saccharomyces cerevisiae and in mouse NIH3T3 cells . The results showed that the frameshifting efficiency was correlated directly to the stem stability between deltaG degrees = -2.5 kcal mol(-1) and deltaG degrees = -19.4 kcal mol(-1) . This demonstrates the essential role of the stability of the stem-loop and does not support the involvement of a specific RNA-binding protein target sequence . However, increasing further the stem stability led to a diminution of frameshifting efficiency, suggesting that the stem-loop acts through a precise kinetic of pausing . Because the same pattern was observed in both yeast and mouse cells, it is likely that the stimulatory mechanism is conserved through evolution. J Biol Chem, 1997 Oct 10, 272(41), 26009 - 16 Heat shock transcription factor 1 binds selectively in vitro to Ku protein and the catalytic subunit of the DNA-dependent protein kinase; Huang J et al.; Heat shock transcription factor 1 (HSF1) functions as the master regulator of the heat shock response in eukaryotes . We have previously shown that, in addition to its role as a transcription factor, HSF1 stimulates the activity of the DNA-dependent protein kinase (DNA-PK) . DNA-PK is composed of two components: a 460-kDa catalytic subunit and a 70- and 86-kDa heterodimeric regulatory component, also known as the Ku protein . We report here that HSF1 binds specifically to each of the two components of DNA-PK . Binding occurs in the absence of DNA . The complex with the Ku protein is stable and forms at a stoichiometry close to unity between the Ku protein heterodimer and the active HSF1 trimer . The binding is blocked by antibodies against HSF1 . Our results show that HSF1 also binds directly, but more weakly, to the catalytic subunit of DNA-PK . Both interactions are dependent on a specific region within the HSF1 regulatory domain . This sequence is necessary but not sufficient for HSF1 stimulation of DNA-PK activity . The ability of HSF1 to interact with both components of DNA-PK provides a potential mechanism for the activation of DNA-PK in response to heat and other forms of stress. J Biol Chem, 1997 Oct 10, 272(41), 25500 - 6 Isolation and characterization of PBP, a protein that interacts with peroxisome proliferator-activated receptor; Zhu Y et al.; In an attempt to identify cofactors that could possibly influence the transcriptional activity of peroxisome proliferator-activated receptors (PPARs), we used a yeast two-hybrid system with Gal4-PPARgamma as bait to screen a mouse liver cDNA library and have identified steroid receptor coactivator-1 (SRC-1) as a PPAR transcriptional coactivator . We now report the isolation of a cDNA encoding a 165-kDa PPARgamma-binding protein, designated PBP which also serves as a coactivator . PBP also binds to PPARalpha, RARalpha, RXR, and TRbeta1, and this binding is increased in the presence of specific ligands . Deletion of the last 12 amino acids from the carboxyl terminus of PPARgamma results in the abolition of interaction between PBP and PPARgamma . PBP modestly increased the transcriptional activity of PPARgamma, and a truncated form of PBP (amino acids 487-735) acted as a dominant-negative repressor, suggesting that PBP is a genuine coactivator for PPAR . In addition, PBP contains two LXXLL signature motifs considered necessary and sufficient for the binding of several coactivators to nuclear receptors . In situ hybridization and Northern analysis showed that PBP is expressed in many tissues of adult mice, including the germinal epithelium of testis, where it appeared most abundant, and during ontogeny, suggesting a possible role for this cofactor in cellular proliferation and differentiation. J Biol Chem, 1997 Oct 10, 272(41), 25455 - 61 Purification and characterization of the alpha-1,3-mannosylmannose-recognizing lectin of Crocus vernus bulbs; Misaki A et al.; A unique mannose-binding lectin, highly specific for terminal Man(alpha1,3)Man groups, was isolated from bulbs of crocus (Crocus vernus All.) . The lectin failed to bind to a mannose affinity column and was purified by simple gel permeation chromatography (Sephacryl S200) . The purified lectin, obtained in crystalline form, had a molecular mass of 44 kDa on gel filtration and showed a single peptide band with a molecular mass of 11 kDa on SDS-polyacrylamide gel electrophoresis, indicating it to be a tetrameric protein composed of four identical subunits . The N-terminal amino acid sequence analysis of the crocus lectin showed essentially no homology with that of other mannose-binding bulb lectins . The crocus lectin selectively interacted with the wild type Saccharomyces cerevisiae and other mannans carrying terminal Man(alpha1,3)Man but not with those lacking this disaccharide unit . In hapten inhibition studies, methyl alpha-mannopyranoside did not inhibit the mannan-lectin interaction . Of various alpha-mannooligosaccharides, those having the Man(alpha1,3)Man sequence showed the highest inhibitory potency, confirming the strict requirement of lectin for terminal alpha1,3-linked mannosylmannose units . An affinity column of immobilized lectin enabled the complete resolution of yeast mannan and glycogen . The immobilized lectin may provide a useful tool for purification and analysis of biologically important polysaccharides and glycoproteins. J Biol Chem, 1997 Oct 10, 272(41), 25413 - 6 COPII subunit interactions in the assembly of the vesicle coat; Shaywitz DA et al.; In vitro analysis of COPII vesicle formation in the yeast Saccharomyces cerevisiae has demonstrated the requirement for three cytosolic factors: Sec31p-Sec13p, Sec23p-Sec24p, and Sar1p . Convergent evidence suggests that the peripheral endoplasmic reticulum (ER) membrane protein Sec16p also represents an important component of the vesicle assembly apparatus: SEC16 interacts genetically with all five COPII genes; Sec16p binds to Sec23p and Sec24p, is found on ER-derived transport vesicles, and is required in vitro for the efficient release of ER-derived vesicle cargo . In this report, we demonstrate an important functional interaction between Sec16p and Sec31p . First, we map onto Sec31p binding regions for Sec16p, Sec23p, Sec24p, and Sec13p . Second, we show that a truncation mutant of Sec31p specifically defective for Sec16p binding is unable to complement a sec31Delta mutant and cannot rescue the secretion defect of a temperature-sensitive sec31 mutant at nonpermissive temperatures . We propose that Sec16p organizes the assembly of a coat that is stabilized both by the interaction of Sec31p with Sec23p and Sec24p and by the interaction of these three components with Sec16p. Plant Physiol, 1997 Sep, 115(1), 283 - 9 Rapid and transient induction of a parsley microsomal delta 12 fatty acid desaturase mRNA by fungal elicitor; Kirsch C et al.; Treatment of cultured parsley (Petroselinum crispum L.) cells with a structurally defined peptide elicitor (Pep25) of fungal origin has previously been shown to cause rapid and large changes in the levels of various desaturated fatty acids . We isolated two distinct parsley cDNAs sharing high sequence similarity with microsomal omega-6 fatty acid desaturases (FADs) . One of them was functionally identified as a delta 12 FAD by expression in the yeast Saccharomyces cerevisiae . Two dienoic fatty acids, hexadecadienoic and linoleic, which were not detectable in control cells, together constituted up to 12% of the total fatty acids in the transformed yeast cells . delta 12 FAD mRNA accumulated rapidly and transiently in elicitor-treated parsley cells, protoplasts, and leaves . These and previous results indicate that fatty acid desaturation is an important early component of the complex defense response of parsley to attempted fungal infection. Radiat Oncol Investig, 1997, 5(4), 163 - 9 Atomic force microscope imaging of DNA and DNA repair proteins: applications in radiobiological research; Pang D et al.; By using the atomic force microscope (AFM), three-dimensional structures of biological specimens may be imaged at nanometer resolution . Furthermore, samples can be imaged in air or in fluid environments . The tapping mode of AFM operation for imaging has offered a significant advance in visualizing soft biological structures, such as DNA, proteins, and membranes . Here, we review the principles underlying the application of this instrument to radiation biological investigations . We focus on examples of proteins involved in the processes of repair of damaged DNA, including poly(ADP-ribose) polymerase, Ku protein, and DNA protein kinase . Novel observations on the character of DNA damage and repair have been addressed by direct visualization of DNA and protein-DNA interactions, such as the observation that the Ku protein is capable of physically joining DNA fragments in vitro . The AFM offers a powerful tool for investigating biologically important molecular interactions that are relevant to DNA damage and repair processes. J Mol Biol, 1997 Oct 3, 272(4), 536 - 40 Two conformations of RNA polymerase II revealed by electron crystallography; Asturias FJ et al.; A new two-dimensional crystal form of yeast RNA polymerase II was obtained in which the conformation of the enzyme appears "open", allowing entry of DNA, as required for the initiation of transcription . By contrast, a previous crystal form contained the enzyme in a "closed" conformation, appropriate for retention of DNA during RNA chain elongation . Interaction with two polymerase subunits, Rpb4 and Rpb7, favors the closed conformation, and binding of general transcription factor TFIIE may do so as well . The effect of Rpb4 and Rpb7, together with previous biochemical evidence, leads to the conclusion that the open to closed transition is a crucial step in the transcription initiation process . Mol Cell Biol, 1997 Nov, 17(11), 6683 - 92 A domain shared by the Polycomb group proteins Scm and ph mediates heterotypic and homotypic interactions; Peterson AJ et al.; The Sex comb on midleg (Scm) and polyhomeotic (ph) proteins are members of the Polycomb group (PcG) of transcriptional repressors . PcG proteins maintain differential patterns of homeotic gene expression during development in Drosophila flies . The Scm and ph proteins share a homology domain with 38% identity over a length of 65 amino acids, termed the SPM domain, that is located at their respective C termini . Using the yeast two-hybrid system and in vitro protein-binding assays, we show that the SPM domain mediates direct interaction between Scm and ph . Binding studies with isolated SPM domains from Scm and ph show that the domain is sufficient for these protein interactions . These studies also show that the Scm-ph and Scm-Scm domain interactions are much stronger than the ph-ph domain interaction, indicating that the isolated domain has intrinsic binding specificity determinants . Analysis of site-directed point mutations identifies residues that are important for SPM domain function . These binding properties, predicted alpha-helical secondary structure, and conservation of hydrophobic residues prompt comparisons of the SPM domain to the helix-loop-helix and leucine zipper domains used for homotypic and heterotypic protein interactions in other transcriptional regulators . In addition to in vitro studies, we show colocalization of the Scm and ph proteins at polytene chromosome sites in vivo . We discuss the possible roles of the SPM domain in the assembly or function of molecular complexes of PcG proteins. Mol Cell Biol, 1997 Nov, 17(11), 6574 - 84 The intermembrane space domain of mitochondrial Tom22 functions as a trans binding site for preproteins with N-terminal targeting sequences; Moczko M et al.; Mitochondrial protein import is thought to involve the sequential interaction of preproteins with binding sites on cis and trans sides of the membranes . For translocation across the outer membrane, preproteins first interact with the cytosolic domains of import receptors (cis) and then are translocated through a general import pore, in a process proposed to involve binding to a trans site on the intermembrane space (IMS) side . Controversial results have been reported for the role of the IMS domain of the essential outer membrane protein Tom22 in formation of the trans site . We show with different mutant mitochondria that a lack of the IMS domain only moderately reduces the direct import of preproteins with N-terminal targeting sequences . The dependence of import on the IMS domain of Tom22 is significantly enhanced by removing the cytosolic domains of import receptors or by performing import in two steps, i.e., accumulation of a preprotein at the outer membrane in the absence of a membrane potential (delta psi) and subsequent import after reestablishment of a delta psi . After the removal of cytosolic receptor domains, two-step import of a cleavable preprotein strictly requires the IMS domain . In contrast, preproteins with internal targeting information do not depend on the IMS domain of Tom22 . We conclude that the negatively charged IMS domain of Tom22 functions as a trans binding site for preproteins with N-terminal targeting sequences, in agreement with the acid chain hypothesis of mitochondrial protein import. Mol Cell Biol, 1997 Nov, 17(11), 6386 - 93 Chromosomal double-strand breaks induce gene conversion at high frequency in mammalian cells; Taghian DG et al.; Double-strand breaks (DSBs) stimulate chromosomal and extrachromosomal recombination and gene targeting . Transcription also stimulates spontaneous recombination by an unknown mechanism . We used Saccharomyces cerevisiae I-SceI to stimulate recombination between neo direct repeats in Chinese hamster ovary (CHO) cell chromosomal DNA . One neo allele was controlled by the dexamethasone-inducible mouse mammary tumor virus promoter and inactivated by an insertion containing an I-SceI site at which DSBs were introduced in vivo . The other neo allele lacked a promoter but carried 12 phenotypically silent single-base mutations that create restriction sites (restriction fragment length polymorphisms) . This system allowed us to generate detailed conversion tract spectra for recipient alleles transcribed at high or low levels . Transient in vivo expression of I-SceI increased homologous recombination 2,000- to 10,000-fold, yielding recombinants at frequencies as high as 1% . Strikingly, 97% of these products arose by gene conversion . Most products had short, bidirectional conversion tracts, and in all cases, donor neo alleles (i.e., those not suffering a DSB) remained unchanged, indicating that conversion was fully nonreciprocal . DSBs in exogenous DNA are usually repaired by end joining requiring little or no homology or by nonconservative homologous recombination (single-strand annealing) . In contrast, we show that chromosomal DSBs are efficiently repaired via conservative homologous recombination, principally gene conversion without associated crossing over . For DSB-induced events, similar recombination frequencies and conversion tract spectra were found under conditions of low and high transcription . Thus, transcription does not further stimulate DSB-induced recombination, nor does it appear to affect the mechanism(s) by which DSBs induce gene conversion. Mol Cell Biol, 1997 Nov, 17(11), 6283 - 93 HOY1, a homeo gene required for hyphal formation in Yarrowia lipolytica; Torres-Guzman JC et al.; The dimorphic fungus Yarrowia lipolytica grows to form hyphae either in rich media or in media with GlcNAc as a carbon source . A visual screening, called FIL (filamentation minus), for Y . lipolytica yeast growth mutants has been developed . The FIL screen was used to identify three Y . lipolytica genes that abolish hypha formation in all media assayed . Y . lipolytica HOY1, a gene whose deletion prevents the yeast-hypha transition both in liquid and solid media, was characterized . HOY1 is predicted to encode a 509-amino-acid protein with a homeodomain homologous to that found in the chicken Hox4.8 gene . Analysis of the protein predicts a nuclear location . These observations suggest that Hoy1p may function as a transcriptional regulatory protein . In disrupted strains, reintroduction of HOY1 restored the capacity for hypha formation . Northern blot hybridization revealed the HOY1 transcript to be approximately 1.6 kb . Expression of this gene was detected when Y . lipolytica grew as a budding yeast, but an increase in its expression was observed by 1 h after cells had been induced to form hyphae . The possible functions of HOY1 in hyphal growth and the uses of the FIL screen to identify morphogenetic regulatory genes from heterologous organisms are discussed. Mol Cell Biol, 1997 Nov, 17(11), 6236 - 45 Elimination of defective alpha-factor pheromone receptors; Jenness DD et al.; This report compares trafficking routes of a plasma membrane protein that was misfolded either during its synthesis or after it had reached the cell surface . A temperature-sensitive mutant form of the yeast alpha-factor pheromone receptor (ste2-3) was found to provide a model substrate for quality control of plasma membrane proteins . We show for the first time that a misfolded membrane protein is recognized at the cell surface and rapidly removed . When the ste2-3 mutant cells were cultured continuously at 34 degrees C, the mutant receptor protein (Ste2-3p) failed to accumulate at the plasma membrane and was degraded with a half-life of 4 min, compared with a half-life of 33 min for wild-type receptor protein (Ste2p) . Degradation of both Ste2-3p and Ste2p required the vacuolar proteolytic activities controlled by the PEP4 gene . At 34 degrees C, Ste2-3p comigrated with glycosylated Ste2p on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that Ste2-3p enters the secretory pathway . Degradation of Ste2-3p did not require delivery to the plasma membrane as the sec1 mutation failed to block rapid turnover . Truncation of the C-terminal cytoplasmic domain of the mutant receptors did not permit accumulation at the plasma membrane; thus, the endocytic signals contained in this domain are unnecessary for intracellular retention . In the pep4 mutant, Ste2-3p accumulated as series of high-molecular-weight species, suggesting a potential role for ubiquitin in the elimination process . When ste2-3 mutant cells were cultured continuously at 22 degrees C, Ste2-3p accumulated in the plasma membrane . When the 22 degrees C culture was shifted to 34 degrees C, Ste2-3p was removed from the plasma membrane and degraded by a PEP4-dependent mechanism with a 24-min half-life; the wild-type Ste2p displayed a 72-min half-life . Thus, structural defects in Ste2-3p synthesized at 34 degrees C are recognized in transit to the plasma membrane, leading to rapid degradation, and Ste2-3p that is preassembled at the plasma membrane is also removed and degraded following a shift to 34 degrees C. Mol Cell Biol, 1997 Nov, 17(11), 6212 - 22 Role for ADA/GCN5 products in antagonizing chromatin-mediated transcriptional repression; Pollard KJ et al.; The Saccharomyces cerevisiae SWI/SNF complex is a 2-MDa multimeric assembly that facilitates transcriptional enhancement by antagonizing chromatin-mediated transcriptional repression . We show here that mutations in ADA2, ADA3, and GCN5, which are believed to encode subunits of a nuclear histone acetyltransferase complex, cause phenotypes strikingly similar to that of swi/snf mutants . ADA2, ADA3, and GCN5 are required for full expression of all SWI/SNF-dependent genes tested, including HO, SUC2, INO1, and Ty elements . Furthermore, mutations in the SIN1 gene, which encodes a nonhistone chromatin component, or mutations in histone H3 or H4 partially alleviate the transcriptional defects caused by ada/gcn5 or swi/snf mutations . We also find that ada2 swi1, ada3 swi1, and gcn5 swi1 double mutants are inviable and that mutations in SIN1 allow viability of these double mutants . We have partially purified three chromatographically distinct GCN5-dependent acetyltransferase activities, and we show that these enzymes can acetylate both histones and Sin1p . We propose a model in which the ADA/GCN5 and SWI/SNF complexes facilitate activator function by acting in concert to disrupt or modify chromatin structure. Arch Biochem Biophys, 1997 Oct 15, 346(2), 269 - 76 Distal site and surface mutations of cytochrome P450 1A2 markedly enhance dehalogenation of chlorinated hydrocarbons; Yanagita K et al.; Chlorinated compounds such as chlorinated ethylenes and ethanes are serious environmental pollutants . In the present study, we examined whether or not a recombinant strain of Saccharomyces cerevisiae that expresses rat liver cytochrome P450 1A2 (P450 1A2) wild-type and mutant proteins can efficiently catalyze oxidative and reductive dehalogenations of trichloroethylene, pentachloroethane, and hexachloroethane . Mutations at putative heme distal and protein surface sites of P450 1A2 greatly enhanced turnover values toward those substrates under both aerobic and anaerobic conditions . For example, a Thr319Ala mutation at the putative heme distal site enhanced the degradation rate of trichloroethylene and pentachloroethane by 2- and 2.7-fold, respectively, under aerobic conditions . The Thr319Ala mutation also strongly facilitated the reaction with hexachloroethane up to 13- and 4.5-fold under aerobic and anaerobic conditions, respectively . The Thr319Ala mutation increased dechlorinated over protonated product ratios by 3-fold as well when either pentachloroethane or hexachloroethane was used as a substrate . A Lys250Leu mutation on the putative protein surface site enhanced the dehalogenation rate of hexachloroethane up to 4.8-fold under anaerobic conditions . In contrast, a Glu318Ala mutation at the putative distal site markedly decreased the activities with trichloroethylene and pentachloroethane substrates under aerobic conditions . Conserved amino acids Thr319 and Glu318 at the heme distal site have been suggested to be important in the O2 activation during monooxidation reactions of P450s . However, the present study indicates that Thr319 is likely to be an inhibitor of dechlorination of trichloroethylene and penta- and hexachloroethanes . The roles of Thr319, Glu318, and Lys250 in the catalysis with chlorinated hydrocarbons are discussed in association with reaction mechanisms. Arch Biochem Biophys, 1997 Oct 15, 346(2), 219 - 29 Nitrobenzimidazoles as substrates for DT-diaphorase and redox cycling compounds: their enzymatic reactions and cytotoxicity; Sarlauskas J et al.; We have synthesized a number of nitrobenzimidazoles containing nitro groups in the benzene ring and found that they acted as relatively efficient substrates for rat liver DT-diaphorase (EC 1.6.99.2), their reactivity exceeding reactivities of nitrofurans and nitrobenzenes . Nitrobenzimidazoles were competitive with NADPH inhibitors of DT-diaphorase in menadione reductase reactions, their inhibition constant being unchanged in the presence of dicumarol and being increased in the presence of 2',5'-ADP . These data indicate that the poor reactivity of nitrobenzimidazoles and other nitroaromatics in comparison to quinones could be determined by their binding in the adenosine-phosphate binding region of the NADPH-binding site, whereas quinones bind at the nicotinamide-binding pocket at the vicinity of FAD of DT-diaphorase . The reduction of 4,5,6-trinitrobenzimidazol-2-one by DT-diaphorase most probably involves reduction of 5-nitro group to 5-nitroso or 5-hydroxylamine derivative at the initial step . A certain parallelism existed between reactivities of nitrobenzimidazoles toward DT-diaphorase and their reactivities in single-electron reduction by Anabaena ferredoxin:NADP+ reductase (EC 1.18.1.2) and Saccharomyces cerevisiae flavocytochrome b2 (EC 1.1.2.3), the latter being determined by electronic factors . However, we suppose that the relatively high reactivity of polinitrobenzimidazoles toward DT-diaphorase was due not only to electronic effects, but also to a sterical crowding of nitrogroups by each other . The toxicity of nitrobenzimidazoles to bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) with a moderate amount of DT-diaphorase (260 U/mg protein) is partly prevented by dicumarol . That points out to partial determination of nitrobenzimidazole cytotoxicity by their reduction by DT-diaphorase . Another important factor of nitrobenzimidazole toxicity to this cell line was oxidative stress, catalyzed by single-electron transferring enzymes. J Virol, 1997 Nov, 71(11), 8552 - 62 Epstein-Barr virus EBNA3C represses Cp, the major promoter for EBNA expression, but has no effect on the promoter of the cell gene CD21; Radkov SA et al.; EBNA3C is a potent repressor of transcription when bound to DNA as a fusion with the DNA binding domain (DBD) of GALA . A survey of promoters has revealed that the wild-type, unfused EBNA3C can specifically repress expression from reporter plasmids containing the Epstein-Barr virus Cp latency-associated promoter . Repression of Cp activity required amino acids 207 to 368, which encompasses a region resembling a basic DBD adjacent to a leucine zipper DNA binding motif and a site which binds to the cellular factor CBF1/RBP-Jkappa . However, amino acids 207 to 368 are dispensable when the protein is bound to DNA as a fusion with the GAL4 DBD, thus implicating this region in DNA binding . Mutation of the CBF1/RBP-Jkappa binding site in EBNA3C abrogated repression, strongly suggesting that CBF1/RBP-Jkappa is necessary for targeting the viral protein to Cp . Consistent with this result, mutation of the EBNA2 response element (a CBF1/RBP-Jkappa binding site) in Cp also prevented significant repression . In addition, amino acids 346 to 543, which were previously defined as important for the repressor activity of the GAL4-EBNA3C fusion proteins, also appear to be necessary for the repression of Cp . Since repression by these fusions was not observed in all cell types, it seems likely that EBNA3C either depends on a corepressor which may interact with amino acids 346 to 543 or is modified in a cell-specific manner in order to repress . These data are consistent with EBNA3C contributing to the regulation of EBNA expression in latently infected B cells through CBF1/RBP-Jkappa and another factor, but this need not directly involve EBNA2 . Finally, although it has been reported that EBNA3C can upregulate CD21 in some B cells, we were unable to demonstrate any effect of EBNA3C on reporter plasmids which contain the CD21 promoter. Proc Natl Acad Sci U S A, 1997 Oct 28, 94(22), 11798 - 802 Prp43: An RNA helicase-like factor involved in spliceosome disassembly; Arenas JE et al.; The Saccharomyces cerevisiae genes PRP2, PRP16, and PRP22 encode pre-mRNA splicing factors that belong to the highly conserved "DEAH" family of putative RNA helicases . We previously identified two additional members of this family, JA1 and JA2 . To investigate its biological function, we cloned the JA1 gene and generated alleles carrying mutations identical to those found in highly conserved regions of other members of the DEAH family . A ja1 allele carrying a mutation identical to that in the temperature-sensitive (ts) prp22-1 gene conferred ts phenotype when integrated into the genome of a wild-type strain by gene replacement . Northern analysis of RNA obtained from the ts strain shifted to a nonpermissive temperature revealed accumulation of unspliced pre-mRNAs and excised intron lariats . Furthermore, analysis of splicing complexes showed that intron lariats accumulated in spliceosomes . The results presented indicate that JA1 encodes a pre-mRNA processing factor (Prp) involved in disassembly of spliceosomes after the release of mature mRNA . We have therefore renamed this gene PRP43. Eur J Biochem, 1997 Sep 15, 248(3), 717 - 23 Palytoxin-induced channel formation within the Na+/K+-ATPase does not require a catalytically active enzyme; Scheiner-Bobis G et al.; It has been demonstrated that palytoxin binds to and forms a channel within the Na+/K+-ATPase . To investigate whether palytoxin-induced channel formation within the sodium pump can occur independently of ATP hydrolysis and phosphorylation of the enzyme, an Asp369-->Ala mutant of the alpha1 subunit of the sheep sodium pump was produced and coexpressed with beta subunits in the yeast Saccharomyces cerevisiae . This aspartic acid residue, which during ion transport becomes phosphorylated from ATP, is essential for the function of the sodium pump . Therefore, as expected, microsomes isolated from yeast expressing the mutant sodium pump do not exhibit any ouabain sensitive ATPase activity, whereas in microsomes from yeast expressing the wild-type sodium pump, 60% of the total ATPase activity is ouabain-sensitive . Ouabain binds to yeast membranes containing either wild-type or mutant sodium pumps with similar Bmax (1.45+/-0.05 versus 1.37+/-0.02 pmol/mg) and Kd values (27.7+/-0.91 versus 29.57+/-0.93 nM), thus indicating that the mutant sodium pumps are expressed in the yeast and that the mutation does not considerably affect the conformation of the enzyme . In the presence of phosphate ouabain binds to microsomes containing the wild-type sodium pump with a Kd of 3.62+/-0.34 nM, showing that, although not necessary, phosphoenzyme formation enhances binding of the steroid . Phosphate or ATP, however, inhibit binding of ouabain to microsomes containing the mutant sodium pump with IC50 values of 78+/-3 microM and 3.0+/-0.4 microM, respectively . Despite these radical changes in the interactions of the mutant enzyme with ouabain, the interactions with palytoxin are not affected by the mutation . Palytoxin causes K+ efflux from yeast cells expressing the wild-type or mutant sodium pumps with EC50 values of 3.5+/-0.4 nM and 6.2+/-0.9 nM, respectively . Palytoxin-induced efflux from cells expressing wild-type or mutant sodium pumps occurs with similar t1/2 values of 20.3+/-2.1 min and 22.2+/-3.1 min, respectively . Ouabain inhibits K+ efflux from both cell types with IC50 values of 28+/-2 microM and 210+/-15 microM, respectively . Cells expressing the Asp369-->Ala mutants have an IC50 7.5-fold higher than that obtained with cells expressing the wild-type sodium pumps, possibly because ATP or phosphate present in the cytosol of the yeast cells influence and decrease ouabain binding to the mutant sodium pump . Thus, while ouabain binding and the associated inhibition of ion fluxes is promoted by phosphorylation of the wild-type enzyme by phosphate or ATP, palytoxin-induced channel formation is independent of phosphorylation and can be separated from the ATPase function of the sodium pump . Since ion fluxes through the sodium pump protein do not depend on ATP hydrolysis, the results suggest that the ionophores of pumps and ion channels might share common structural features. Mol Gen Genet, 1997 Sep, 256(1), 18 - 27 Trichoderma reesei sequences that bind to the nuclear matrix enhance transformation frequency; Belshaw NJ et al.; Three DNA fragments, trs1, 2 and 3, were isolated from the Trichoderma reesei genome on the basis of their ability to promote autonomous replication of plasmids in Saccharomyces cerevisiae . Each trs element bound specifically to the isolated T . reesei nuclear matrix in vitro, and two of them bound in vivo, indicating that they are matrix attachment regions (MARs) . A similar sequence previously isolated from Aspergillus nidulans (ans1) was also shown to bind specifically to the T . reesei nuclear matrix in vitro . The T . reesei MARs are AT-rich sequences containing 70%, 86% and 73% A + T over 2.9, 0.8 and 3.7 kb, respectively for trs1, 2 and 3 . They exhibited no significant sequence homology, but were shown to contain a number of sequence motifs that occur frequently in many MARs identified in other eukaryotes . However, these motifs occurred as frequently in the trs elements as in randomly generated sequences with the same A + T content . trs1 and 3 were shown to be present as single copies in the T . reesei genome . The presence of the trs elements in transforming plasmids enhanced the frequency of integrative transformation of T . reesei up to five fold over plasmids without a trs . No evidence was obtained to suggest that the trs elements promoted efficient replication of plasmids in T . reseei . A mechanism for the enhancement of transformation frequency by the trs elements is proposed. J Biol Chem, 1997 Oct 24, 272(43), 27444 - 9 Identification of elongin C sequences required for interaction with the von Hippel-Lindau tumor suppressor protein; Takagi Y et al.; Elongin C is a 112-amino acid protein that is found in mammalian cells as a positive regulatory subunit of heterotrimeric RNA polymerase II elongation factor Elongin (SIII) and as a component of a multiprotein complex containing the von Hippel-Lindau (VHL) tumor suppressor protein . As a subunit of the Elongin complex, Elongin C interacts directly with the transcriptionally active Elongin A subunit and potently induces its elongation activity; in addition, Elongin C interacts with the ubiquitin-like Elongin B subunit, which regulates the interaction of Elongin C with Elongin A . As a component of the VHL complex, Elongin C interacts directly with both Elongin B and the VHL protein . Binding of the VHL protein to Elongin C was found to prevent Elongin C from interacting with and activating Elongin A in vitro, leading to the proposal that one function of the VHL protein may be to regulate RNA polymerase II elongation by negatively regulating the Elongin complex . In this report, we identify Elongin C sequences required for its interaction with the VHL protein . We previously demonstrated that the ability of Elongin C to bind and activate Elongin A is sensitive to mutations in the C-terminal half of Elongin C, as well as to mutations in an N-terminal Elongin C region needed for formation of the Elongin BC complex . Here we show that interaction of Elongin C with the VHL tumor suppressor protein depends strongly on sequences in the C terminus of Elongin C but is independent of the N-terminal Elongin C region required for binding to Elongin B and for binding and activation of Elongin A . Taken together, our results are consistent with the proposal that the VHL protein negatively regulates Elongin C activation of the Elongin complex by sterically blocking the interaction of C-terminal Elongin C sequences with Elongin A . In addition, our finding that only a subset of Elongin C sequences required for its interaction with Elongin A are critical for binding to VHL may offer the opportunity to develop reagents that selectively interfere with Elongin and VHL function. J Biol Chem, 1997 Oct 24, 272(43), 27281 - 7 Serine phosphorylation-dependent association of the band 4.1-related protein-tyrosine phosphatase PTPH1 with 14-3-3beta protein; Zhang SH et al.; PTPH1 is a human protein-tyrosine phosphatase with homology to the band 4.1 superfamily of cytoskeletal-associated proteins . PTPH1 was found to associate with 14-3-3beta using a yeast two-hybrid screen, and its interaction could be reconstituted in vitro using recombinant proteins . Examination of the interaction between 14-3-3beta and various deletion mutants of PTPH1 by two-hybrid tests suggested that the integrity of the PTP is important for this binding . Although both PTPH1 and Raf-1 form complexes with 14-3-3beta, they appear to do so independently . Binding of 14-3-3beta to PTPH1 in vitro was abolished by pretreating PTPH1 with potato acid phosphatase and was greatly enhanced by pretreating with Cdc25C-associated protein kinase . Thus the association between PTPH1 and 14-3-3beta is phosphorylation-dependent . Two novel motifs RSLS359VE and RVDS853EP in PTPH1 were identified as major 14-3-3beta-binding sites, both of which are distinct from the consensus binding motif RSXSXP recently found in Raf-1 . Mutation of Ser359 and Ser853 to alanine significantly reduced the association between 14-3-3beta and PTPH1 . Furthermore, association of PTPH1 and 14-3-3beta was detected in several cell lines and was regulated in response to extracellular signals . These results raise the possibility that 14-3-3beta may function as an adaptor molecule in the regulation of PTPH1 and may provide a link between serine/threonine and tyrosine phosphorylation-dependent signaling pathways. J Biol Chem, 1997 Oct 24, 272(43), 27259 - 65 Defining the minimal domain of Ku80 for interaction with Ku70; Osipovich O et al.; The Ku protein has a critical function in the repair of double-strand DNA breaks induced for example by ionizing radiation or during VDJ recombination . Ku serves as the DNA-binding subunit of the DNA-dependent kinase and is a heterodimeric protein composed of 80- and 70-kDa subunits . We used the two-hybrid system to analyze the interaction domains of the Ku subunits and to identify possible additional partners for Ku . Screening a human cDNA library with the Ku heterodimer did not reveal any novel partners . Screening with the individual subunits, we detected only Ku70 clones interacting with Ku80 and only Ku80 clones interacting with Ku70, indicating that these are the primary partners for one another . Ku80 and Ku70 formed only heterodimers and did not homodimerize . Ku80 was restricted to interacting with just one Ku70 molecule at a time . The minimal functional interaction domain of Ku80 that interacted with Ku70 was defined . It consisted of a 28-amino acid region extending from amino acid 449 to 477 . This region was crucial for interaction with Ku70, since mutation within this critical site at amino acids 453 and 454 abrogated the ability to interact with Ku70 . We furthermore verified that the same region is crucial for interaction with Ku70 using in vitro co-translation of both subunits followed by an immunoprecipitation with anti-Ku70 antibodies . This interaction domain of Ku80 does not contain any motif previously recognized in protein-protein interactions. J Biol Chem, 1997 Oct 24, 272(43), 27253 - 8 The histone acetyltransferase activity of human GCN5 and PCAF is stabilized by coenzymes; Herrera JE et al.; Here we report that PCAF and human GCN5, two related type A histone acetyltransferases, are unstable enzymes that under the commonly used assay conditions are rapidly and irreversibly inactivated . In addition, we report that free histone H1, although not acetylated in vivo, is a preferred and convenient in vitro substrate for the study of PCAF, human GCN5, and possibly other type A histone acetyltransferases . Using either histone H1 or histone H3 as substrates, we find that preincubation with either acetyl-CoA or CoA stabilizes the acetyltransferase activities of PCAF, human GCN5 and an enzymatically active PCAF deletion mutant containing the C-terminal half of the protein . The stabilization requires the continuous presence of coenzyme, suggesting that the acetyltransferase-coenzyme complexes are stable, while the isolated apoenzymes are not . Human GCN5 and the N-terminal deletion mutant of PCAF are stabilized equally well by preincubation with either CoA or acetyl-CoA, while intact PCAF is better stabilized by acetyl-CoA than by CoA . Intact PCAF, but not the N-terminal truncation mutant or human GCN5, is autoacetylated . These findings raise the possibility that the intracellular concentrations of the coenzymes affect the stability and therefore the nuclear activity of these acetyltransferases. J Biol Chem, 1997 Oct 24, 272(43), 27160 - 6 Functional domain mapping of the clathrin-associated adaptor medium chains mu1 and mu2; Aguilar RC et al.; The clathrin-associated adaptors AP-1 and AP-2 are heterotetrameric complexes involved in the recognition of sorting signals present within the cytosolic domain of integral membrane proteins . The medium chains of these complexes, mu1 and mu2, have been implicated in two types of interaction: assembly with the beta1 and beta2 chains of the corresponding complexes and recognition of tyrosine-based sorting signals . In this study, we report the results of a structure-function analysis of the mu1 and mu2 chains aimed at identifying regions of the molecules that are responsible for each of the two interactions . Analyses using the yeast two-hybrid system and proteolytic digestion experiments suggest that mu1 and mu2 have a bipartite structure, with the amino-terminal one-third (residues 1-145 of mu1 and mu2) being involved in assembly with the beta chains and the carboxyl-terminal two-thirds (residues 147-423 of mu1 and 164-435 of mu2) binding tyrosine-based sorting signals . These observations support a model in which the amino-terminal one-third of mu2 is embedded within the core of the AP-2 complex, while the carboxyl-terminal two-thirds of the protein are exposed to the medium, placing this region in a position to interact with tyrosine-based sorting signals. J Biol Chem, 1997 Oct 24, 272(43), 26991 - 8 Isolation and characterization of a dual prenylated Rab and VAMP2 receptor; Martincic I et al.; Rab GTPases have been implicated in intracellular vesicle trafficking . Using the yeast two-hybrid screen, we have isolate